JP6188760B2 - Modified toxin - Google Patents

Description

The present invention relates to a modified toxin, a fusion protein containing the modified toxin, a composition thereof, a method for preparing the modified toxin, and a method for treating diseases such as cancer using the modified toxin.

(Cross-reference)
This application is based on US Provisional Patent Application No. 60 / 945,556 filed on June 21, 2007 (Attorney Docket No. 33094-702101), and U.S. Provisional Patent Application filed on August 6, 2007. 60 / 954,278 (Agent reference number 33094-702102), 2
US Provisional Patent Application No. 61 / 032,888 filed on Feb. 29, 2008 (Attorney Docket No. 33094-702103), US Provisional Patent Application No. 61 filed Apr. 3, 2008
/ 042,178 (Attorney Docket No. 33094-70204), June 2, 2007
No. 60 / 945,568 (Attorney Docket No. 33094-)
703.101), U.S. Provisional Application No. 60 / 954,28 filed Aug. 6, 2007.
No. 4 (Attorney Docket Number 33094-703.102), U.S. Provisional Patent Application No. 61 / 032,910 filed on Feb. 29, 2008 (Attorney Docket Number 33094-703.103)
) And US Provisional Application No. 61 / 042,187 (Attorney Docket No. 33094-703.104) filed Apr. 3, 2008. Each of these applications is
Which is incorporated herein by reference in its entirety.

The effect of a therapeutic protein can be limited, for example, by an unwanted immune response to the therapeutic protein. For example, some mouse monoclonal antibodies have shown promise as a therapy in many human disease settings, but in some cases have become nonfunctional due to the induction of a significant degree of human anti-mouse antibody (HAMA) response. [Schroff, R.A. W.
et al. (1985) Cancer Res. 45: 879-885; Shawl
er, D.C. L. et al. (1985) J. Am. Immunol. 135: 1530-1
535]. A number of techniques have been developed for monoclonal antibodies in an attempt to reduce the HAMA response [WO 89/09622;
P0239400; European Patent Application Publication No. EP0438310; International Publication No. WO
91/06667]. These recombinant DNA approaches generally reduce mouse genetic information in the final antibody structure while increasing human genetic information in the final structure. Nevertheless, the resulting “humanized” antibodies still elicit an immune response in patients in some cases [Issacs J. et al. D. (1990) S
em. Immunol. 2: 449, 456; Rebello, P .; R. et al.
(1999) Transplantation 68: 1417-1420].

Antibodies are not the only type of polypeptide molecule that is administered as a therapeutic that can initiate an immune response. Proteins of human origin and proteins having the same amino acid sequence as those occurring internally in humans can further elicit an immune response in humans. A notable example is granulocyte macrophage colony stimulating factor [Wadwa, M .; et
al. (1999) Clin. Cancer Res. , 5: 1353-1361] and interferon α2 [Russo, D. et al. et al. , (1996) Bri. J. et al. H
aem. 94: 300-305; Stein, R .; et al. , (1988) New
Engl. J. et al. Med. , 318: 1409-1413]. In such situations where these human proteins are immunogenic, there is a presumed failure of immunological tolerance to these proteins that are otherwise functioning in these subjects.

A sustained antibody production response to therapeutic proteins requires stimulation of helper T cell proliferation and activation. T cell stimulation requires interaction between T cells and antigen presenting cells (APCs). At the heart of the interaction is the T cell receptor (TCR) on the surface of the T cell that is involved in the peptide MHC class II complex on the surface of APC. Peptides are derived from intracellular processing of antigenic proteins. Peptide sequences derived from protein antigens that can stimulate T cell activity by presentation on the surface of MHC class II molecules are commonly referred to as “T cell epitopes”. Such T cell epitopes are any amino acid residue sequence that has the ability to bind to MHC class II molecules and, at least in principle, these T cells by engaging the TCR in promoting a T cell response. Activation can occur. For many proteins, a small number of helper T cell epitopes drive T-helper signaling and have a sustained high affinity for what can be an exposed surface determinant of a very broad repertoire of therapeutic protein surfaces It is understood that this can result in a class-converting antibody response.

Identification of T cell epitopes is recognized as the first step towards epitope elimination of T cell epitopes in therapeutic proteins. International Publication No. WO 98/52976 and W
O00 / 34317 teaches a computational threading method for identifying polypeptide sequences that have the potential to bind a subset of human MHC class II DR allotypes. In these applications, predicted T cell epitopes are identified by calculation and subsequently removed by the use of careful amino acid substitutions within the protein of interest. However, epitope identification methods based on this scheme and other calculations [Godkin, A. et al. J. et al. et
al. (1998) J. MoI. Immunol. 161: 850-858;
olo, T .; et al. (1999) Nat. Biotechnol. , 17:55
5-561], peptides that are predicted to be able to bind MHC class II molecules function as T cell epitopes in all situations, especially in vivo, due to processing pathways or other phenomena. It has been found that it cannot be obtained. Furthermore, the computational methods for T cell epitope prediction were generally unable to predict epitopes with DP or DQ restrictions.

For example, in vitro methods for measuring the ability of synthetic peptides to bind MHC class II molecules using the MHC allotype B cell line defined as a source of MHC class II binding surfaces [Ma
rshall K.M. W. et al. (1994) J. Am. Immunol. , 152: 4
946-4956; O'Sullivan et al. (1990) J. MoI. Immun
ol. 145: 1799-1808; Robadey C., et al. et al. , (1997
) J. et al. Immunol. , 159: 3238-3246] can be applied to the identification of MHC class II ligands. However, such techniques are not amenable to screening a large number of possible epitopes against a wide variety of MHC allotypes and cannot confirm the ability of the binding peptide to function as a T cell epitope.

In addition to T cell epitopes, a number of proteins are known to induce vascular leakage syndrome (VLS). VLS results from protein-mediated damage to the vascular endothelium. In the case of recombinant proteins, immunotoxins and fusion toxins, the damage is initiated by the interaction between the therapeutic protein and vascular endothelial cells.

The underlying mechanism of VLS is unclear, possibly with a cascade of events initiated in endothelial cells (EC), and with an inflammatory cascade and cytokines (Engert et al., 1997). VLS has a complex etiology that includes damage to vascular endothelial cells (EC) and fluid and protein spills leading to interstitial edema, weight gain and its most severe forms of kidney damage, aphasia and pulmonary edema (Sausville)
and Vitetta, 1997; Baluna and Vitetta, 1996
Engert et al. , 1997).

One of the VLS motifs found in ricin toxin, the “LDV” motif, has been reported to essentially mimic the activity of the fibronectin subdomain required to bind to integrin receptors. Integrins mediate cell-cell and cell-extracellular matrix interactions (ECM). Integrins function as receptors for various cell surface and extracellular matrix proteins such as fibronectin, laminin, vitronectin, collagen, osteospondin, thrombospondin and von Willebrand factor. Integrins play an important role in the development and maintenance of the vasculature and affect endothelial cell adhesion during angiogenesis. In addition, a ricin “LDV” motif can be found in rotavirus coat protein, and this motif has been reported to be important for virus binding and entry into cells (Coulson, et al., P.).
roc. Natl. Acad. Sci. USA, 94 (10): 5389-5494 (1
997)). Thus, endothelial cell adhesion, vascular stability, and human vascular endothelial cells (HUVEC
There appears to be a direct link between lysine binding to) and the VLS motif that mediates vascular leakage.

Mutant deglycosylated ricin toxin A chain (dgRTA) in which this motif has been removed by conservative amino acid substitutions has been assembled, and these mutants have demonstrated smaller VLS effects in a mouse model (Smallshaw et al ., Nat.
Biotechnol. , 21 (4): 387-91 (2003)). However, most of these structures produce dgRTA mutants that were not as cytotoxic as wild-type ricin toxins, and mutations produced important and functionally important structural changes in ricin toxins. It suggests that. It should also be noted that no evidence was obtained suggesting that motifs in dgRTA mediated VLS in HUVEC interactions and other proteins. Various studies have revealed that the majority of mutant dgRTAs are less effective carriers and suggest that these mutant toxins can be used to assemble fusion toxins. There was no evidence to do.

VLS is often observed during bacterial sepsis and may involve IL-2 and various other cytokines (Baluna and Vitetta, J. Immunothee).
r. (1999) 22 (1): 41-47). VLS is also observed in patients receiving protein fusion toxins or recombinant cytokine therapy. VLS can manifest as hypoalbuminemia, weight gain, pulmonary edema and hypotension. In some patients receiving immunotoxins and fusion toxins, myalgia and rhabdomyolysis result from VLS as a function of fluid accumulation in muscle tissue or brain microvasculature (Smallshaw et al
. Nat. Biotechnol. , 21 (4): 387-91 (2003)). VL
S occurs in patients treated with immunotoxins containing ricin A chain, saporin, Pseudomonas exotoxin A and diphtheria toxin (DT). All clinical trials regarding the usefulness of targeted toxins, immunotoxins and recombinant cytokines reported that VLS effects and VLS-like effects were observed in the treatment population. VLS occurred in approximately 30% of patients treated with DAB ₃₈₉ IL-2 (Foss et al., Clin. Lym).
poma, 1 (4): 298-302 (2001), Figgitt et al. ,
Am. J. et al. Clin. Dermatol. 1 (1): 67-72 (2000)). DA
B ₃₈₉ IL-2 (also referred to in the present application as DT ₃₈₇ -IL2 in the same meaning) is the catalytic (C) domain of DT (DT poison) that is gene-fused to interleukin 2 (IL-2) as a target ligand and A protein fusion toxin consisting of a transmembrane (T) domain. [Wil
liams et al. , Protein Eng. 1: 493-498 (1987
); Williams et al. , J .; Biol. Chem. , 265: 11885.
-11889 (1990); Williams et al. , J .; Biol. Chem
. , 265 (33): 20673-20877; Waters et al. , Ann.
New York Acad. Sci. , 30 (636): 403-405, (1991
); Kiyokawa et al. , Protein Engineering, 4 (
4): 463-468 (1991); Murphy et al. , In Handbo
ok of Experimental Pharmacology, 145: 91-1.
04 (2000)].

VLS has also been observed after administration of IL-2, growth factors, monoclonal antibodies and traditional chemotherapy. Severe VLS can cause fluid and protein extravasation, edema, reduced tissue perfusion, withdrawal of therapy and organ failure [Vitetta et al. ,
Immunology Today, 14: 252-259 (1993); Siegal
l et al. , Proc. Natl. Acad. Sci. , 91 (20): 9514
-9518 (1994); Baluna et al. , Int. J. et al. Immunoph
armalogy, 18 (6-7): 355-361 (1996); Baluna
et al. , Immunopharmacology, 37 (2-3): 117-13.
2 (1997); Bascon, Immunopharmacology, 39 (3):
255 (1998)].

International Publication No. WO89 / 09622 Specification European Patent Application Publication No. EP0239400 European Patent Application Publication No. EP 0438310 International Publication No. WO91 / 06667 Specification

Schroff, R.M. W. et al. (1985) Cancer Res. 45: 879-885 Shawler, D .; L. et al. (1985) J. Am. Immunol. 135: 1530-1535 Issacs J.M. D. (1990) Sem. Immunol. , 2: 449, 456 Rebello, P.M. R. et al. (1999) Transplantation 68: 1417-1420. Wadhwa, M .; et al. (1999) Clin. Cancer Res. , 5: 1353-1361 Russo, D.C. et al. , (1996) Bri. J. et al. Haem. 94: 300-305 Stein, R.M. et al. (1988) New Engl. J. et al. Med. 318: 1409-1413. Godkin, A .; J. et al. et al. (1998) J. MoI. Immunol. 161: 850-858 Sturniolo, T .; et al. (1999) Nat. Biotechnol. , 17: 555-561 Marshall K.M. W et al. (1994) J. Am. Immunol. , 152: 4946-4756 O'Sullivan et al. (1990) J. MoI. Immunol. 145: 1799-1808. Robadey C.I. et al. (1997) J. MoI. Immunol. 159: 3238-3246 Coulson, et al. , Proc. Natl. Acad. Sci. USA, 94 (10): 5389-5494 (1997). Smallshaw et al. Nat. Biotechnol. , 21 (4): 387-91 (2003) Baluna and Vitetta, J.A. Immunother. , (1999) 22 (1): 41-47. Foss et al. , Clin. Lymphoma, 1 (4): 298-302 (2001) Figgitt et al. , Am. J. et al. Clin. Dermatol. , 1 (1): 67-72 (2000) Williams et al. , Protein Eng. 1: 493-498 (1987) Williams et al. , J .; Biol. Chem. , 265: 11885-11889 (1990). Williams et al. , J .; Biol. Chem. , 265 (33): 20673-20677. Waters et al. , Ann. New York Acad. Sci. , 30 (636): 403-405, (1991) Kiyokawa et al. , Protein Engineering, 4 (4): 463-468 (1991). Murphy et al. , In Handbook of Experimental Pharmacology, 145: 91-104 (2000). Vitetta et al. , Immunology Today, 14: 252-259 (1993). Siegall et al. , Proc. Natl. Acad. Sci. , 91 (20): 9514-9518 (1994). Baluna et al. , Int. J. et al. Immunopharmacology, 18 (6-7): 355-361 (1996). Baluna et al. , Immunopharmacology, 37 (2-3): 117-132 (1997). Bascon, Immunopharmacology, 39 (3): 255 (1998)

Accordingly, there is a need to design modified diphtheria toxins that result in a decrease in vascular leakage syndrome compared to wild type diphtheria toxin and / or have reduced immunogenicity compared to wild type diphtheria toxin.

Provided herein are modified toxins, fusion proteins containing modified toxins, compositions thereof, methods of preparing modified toxins, and methods of treating diseases such as cancer using modified toxins.

A modified toxin comprising a toxin having at least one amino acid residue modification in at least one T cell epitope, wherein the modified toxin exhibits reduced immunogenicity compared to an unmodified toxin Provided herein are fusion proteins containing modified toxins, compositions thereof, methods of preparing modified toxins and methods of treating diseases.

Provided herein are modified diphtheria toxins that contain at least one amino acid residue modification in at least one T cell epitope. The modified toxin exhibits reduced immunogenicity compared to the unmodified toxin. T cell epitopes are SEQ ID NO: 181, 184, 187, 1
An amino acid sequence selected from 90, 193, 196 and 199 can be contained.
In one embodiment of the compounds disclosed herein, the at least one amino acid residue modification of the modified diphtheria toxin is SEQ ID NO: 181, 184, 187, 190, 193, 1
Performed with 96 and 199 epitope cores. In yet another embodiment, the at least one amino acid residue modification is SEQ ID NO: 181, 184, 187, 190, 193, 19
Performed at the N-terminus, C-terminus, or both of 6 and 199. In yet another embodiment, the at least one amino acid residue modification is SEQ ID NO: 181, 184, 187, 190,
Performed at the epitope core of 193, 196 and 199 and at the N-terminus, C-terminus, or both.

In one embodiment, the modified diphtheria toxin is V7S, V7T, V7N, V7D,
D8E, S9A, S9T, V29S, V29T, V29N, V29D, D30E, S31
N, I290T, D291E, S292A, S292T, V97A, V97T, V97D
, L107N, M116A, M116Q, M116N, F124H, V148A, V14
Contains one or more modifications selected from 8T, L298A and L298N.

In one embodiment, the modified diphtheria toxin is V7N, V7T, V29N, V29.
Contains two modifications selected from among T and V29D. Modifications include, but are not limited to, V7N V29N, V7N V29T, V7N V29D, V7T V29N
, V7T V29T or V7T V29D.

In one embodiment, the modified diphtheria toxin is V7D, V7N, V7T, L107.
Contains three modifications selected from A, L107N and F124H. Modifications include, but are not limited to, V7D L107A F124H, V7D L107N F1
24H, V7N L107A F124H, V7N L107N F124H, V7T
L107A F124H and V7T L107N F124H.

In one embodiment, the DT variant is, for example, V7D V97D L107N.
F124H, V7N V97D L107N F124H, V7T V97A L10
7N F124H, V7T V97D L107N F124H, V7T V97TL
107N F124H, V7D V97A L107N F124H, V7D V97T
L107N F124H, V7N V97A L107N F124H and V7N V
Contains four modifications such as 97T L107N F124H.

Provided herein are modified toxins (including fusions containing toxins), wherein the toxin consists of diphtheria toxin or a fragment thereof and exhibits reduced immunogenicity compared to unmodified diphtheria toxin. The Further provided herein are modified toxins that exhibit reduced immunogenicity that contain diphtheria toxin and at least one cell binding domain of a ligand derived from a non-diphtheria toxin polypeptide. In one embodiment, the cell binding domain derived from a non-diphtheria toxin polypeptide is a cell binding ligand. In another embodiment of the compounds disclosed herein, the modified toxin is a fusion toxin and the non-toxin polypeptide is a cell binding ligand, such as but not limited to an antibody or antigen binding fragment thereof, cytokine, poly A fusion toxin that is a peptide, hormone, growth factor or insulin.

In one embodiment, the modified toxin is a fusion toxin, wherein the cell binding domain is an antibody or antigen binding fragment thereof. The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a genetically modified antibody, or a transplanted antibody. The antigen-binding fragment can be, for example, Fab, Fab ₂ , F (ab ′) ₂ , scFv, scFv ₂ , single chain binding polypeptide, V _H or V _L. In another embodiment, the antibody or antigen-binding fragment thereof binds to a B cell surface molecule such as, for example, B cell surface molecule CD19 or CD22. Alternatively, the antibody or antigen-binding fragment thereof binds to the ovarian receptor MISIIR (Mueller inhibitor type II receptor).

Non-diphtheria toxin polypeptides include, but are not limited to, antibodies or antigen-binding fragments thereof, EGF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, INFα, INFγ, GM-CSF, G-CSF, M-CSF, TNF, V
It can consist of EGF, ephrin, BFGF and TGF. In one embodiment, the cytokine is IL2.

A modified toxin comprising a toxin having at least one amino acid residue modification in at least one T cell epitope, wherein the modified toxin exhibits reduced immunogenicity as compared to an unmodified toxin. Such modified toxins are provided herein. Furthermore, there is provided a modified toxin having at least one amino acid residue modification in at least one T cell epitope, wherein the toxin is diphtheria toxin or a fragment thereof. Also provided is a modified toxin having at least one amino acid residue modification in at least one T cell epitope, wherein the toxin is a diphtheria fusion toxin. Furthermore, a modified toxin having at least one amino acid residue modification in at least one T cell epitope, the toxin comprising diphtheria toxin and at least one cell binding main derived from a non-diphtheria toxin polypeptide. A modified toxin characterized in that it is a diphtheria fusion toxin. A modified toxin having at least one amino acid residue modification in at least one T cell epitope, wherein the toxin comprises diphtheria toxin and at least one cell-binding main derived from a non-diphtheria toxin polypeptide. Provided is a modified toxin characterized in that it is a diphtheria fusion toxin and the non-diphtheria toxin polypeptide is IL-2.

A composition comprising a modified diphtheria toxin having reduced immunogenicity and reduced binding to endothelial cells, wherein the modified diphtheria toxin is an amino acid sequence as listed in SEQ ID NO: 2 or 200, Containing an amino acid sequence having one or more amino acid modifications therein, SEQ ID NOs: 181, 184, 187, 190, 193, 196 and 199
Wherein at least one T cell epitope containing an amino acid sequence selected from is modified, and at least one amino acid modification is a residue 7-9, 29- of SEQ ID NO: 2 or 200
Performed within a (x) D / E (y) motif in a region selected from 31 and 290-292, and the modified diphtheria toxin has cytotoxicity comparable to unmodified diphtheria toxin;
Modifications at position (x) are A, S, E, F, C, M, T, W, Y, P, H, Q, D, N, K
, R, G, L and V by amino acid residues selected from the modified or unusual amino acids in Table 1
Or a substitution of I; and / or modification at position D / E is A, S, E, I, V, L, F,
C, M, G, T, W, Y, P, H, Q, N, K, R and substitution of D or E with amino acid residues selected from among the modified or unusual amino acids of Table 1; and / Or modification at position (y) is I, F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K, R, S, L
A modified diphtheria toxin having reduced immunogenicity and reduced binding to endothelial cells characterized by being a substitution with an amino acid residue selected from among the modified or unusual amino acids of Table 1, and V Compositions are provided herein.

The unmodified diphtheria toxin can have, for example, the amino acid sequence of SEQ ID NO: 2 or 200, or any one amino acid sequence of SEQ ID NO: 4-147.

In one embodiment, the modified diphtheria toxin is V7T, V7N, V7D, D8N,
S9A, S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31
Contains one or more modifications selected from N, I290T, D291E, S292A, S292G and S292T.

In one embodiment, the modified diphtheria toxin contains two modifications. Such modified diphtheria toxins are, for example, V7N V29N, V7N V29T, V7N V29.
Combinations of mutations such as D, V7T V29N, V7T V29T or V7T V29D can be included.

In one embodiment, the modified diphtheria toxin contains three modifications. Such modified diphtheria toxins are, for example, V7N V29N I290N, V7N V29N I
290T, V7N V29N S292A, V7N V29N S292T, V7N V
29T I290N, V7N V29T I290T, V7N V29T S292A,
Combinations of mutations such as V7N V29T S292T and V7T V29T I290T can be included.

Compositions containing modified diphtheria toxin have reduced immunogenicity (modified T cell epitope and / or B cell epitope) and human vascular endothelial cells (HU) compared to unmodified diphtheria toxin.
(Has) reduced binding activity against (VEC). Such compositions include non-diphtheria toxin polypeptides such as, but not limited to, antibodies or antigen-binding fragments thereof, EGF
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8
IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-1
5, INFα, INFγ, GM-CSF, G-CSF, M-CSF, TNF, VEGF,
Ephrin, BFGF and TGF can further be contained. A non-diphtheria toxin polypeptide can also be a fragment of such a polypeptide, eg, a cell binding portion thereof. In one embodiment, the non-diphtheria toxin polypeptide is IL-2 or a cell binding portion thereof.

A modified diphtheria toxin comprising: (i) identifying at least one T cell epitope within the diphtheria toxin; and (ii) at least one amino acid residue within at least one T cell epitope identified in (i) A modified diphtheria toxin prepared by a method for modifying a group, wherein the modified diphtheria toxin exhibits reduced immunogenicity compared to an unmodified toxin,
Provided herein.

A method of selecting a modified diphtheria toxin that exhibits reduced immunogenicity and reduced binding to endothelial cells compared to an unmodified toxin, comprising: (i) at least within the amino acid sequence of the diphtheria toxin of SEQ ID NO: 2 or 200 Identifying one T cell epitope; (ii) modifying at least one amino acid residue within at least one T cell epitope identified in (i); and (iii) remaining SEQ ID NO: 2 or 200 Modify at least one amino acid residue in a region selected from the group consisting of groups 7-9, 29-31 and 290-292, wherein the modified diphtheria toxin is reduced compared to the unmodified toxin And (iv) analyzing the modified amino acid sequence of the modified diphtheria toxin to determine at least one T cell epitope Consists of confirming that at least one VLS motif is modified, and (v) selecting a modified diphtheria toxin that exhibits reduced immunogenicity and reduced binding to endothelial cells compared to the unmodified toxin A method is provided herein.

A method of preparing a modified diphtheria toxin exhibiting reduced immunogenicity compared to an unmodified toxin comprising: (i) identifying at least one T cell epitope within the amino acid sequence of the diphtheria toxin of SEQ ID NO: 2 or 200. , (Ii) A method comprising modifying at least one amino acid residue within at least one T cell epitope identified in (i) is provided herein.

A method of preparing a modified diphtheria toxin exhibiting reduced immunogenicity and reduced binding to endothelial cells compared to an unmodified toxin, comprising: (i) at least one within the amino acid sequence of the diphtheria toxin of SEQ ID NO: 2 or 200 (Ii) modifying at least one amino acid residue within at least one T cell epitope identified in step (i); and (iii) the remainder of SEQ ID NO: 2 or 200 Groups 7-9, 29-31 and 290-292
Modifying at least one amino acid residue in a region selected from the group consisting of: said modified diphtheria toxin exhibits reduced immunogenicity and reduced binding to endothelial cells compared to unmodified toxin A method characterized by is further provided herein.

A method of preparing a modified diphtheria toxin exhibiting reduced immunogenicity and reduced binding to endothelial cells compared to an unmodified toxin, comprising: (i) at least one within the amino acid sequence of the diphtheria toxin of SEQ ID NO: 2 or 200 (Ii) modifying at least one amino acid residue within at least one T cell epitope identified in (i);
ii) modify at least one amino acid residue in a region selected from the group consisting of residues 7-9, 29-31 and 290-292 of SEQ ID NO: 2 or 200; (iv) modification of a modified diphtheria toxin Analyzing the determined amino acid sequence to identify whether the modification of the T cell epitope results in a VLS motif, and (v) modifying the VLS motif identified in (iv) Further provided herein are methods characterized in that diphtheria toxin exhibits reduced immunogenicity and decreased binding to endothelial cells compared to unmodified toxin.

A method of preparing a modified diphtheria toxin exhibiting reduced immunogenicity and reduced binding to endothelial cells compared to an unmodified toxin, comprising: (i) at least one within the amino acid sequence of the diphtheria toxin of SEQ ID NO: 2 or 200 (Ii) modifying at least one amino acid residue within at least one T cell epitope identified in (i);
ii) modify at least one amino acid residue in a region selected from the group consisting of residues 7-9, 29-31 and 290-292 of SEQ ID NO: 2 or 200; (iv) modification of a modified diphtheria toxin Analyzing whether the VLS motif modification results in a T cell epitope, and (v) modifying the T cell epitope identified in (iv), Further provided herein is a method characterized in that the modified diphtheria toxin exhibits reduced immunogenicity and reduced binding to endothelial cells compared to the unmodified toxin.

A method for selecting a modified diphtheria toxin, wherein the modified diphtheria toxin has lost at least one B cell epitope, comprising: (i) serum from a immunized subject with a diphtheria toxin vaccine A sample is obtained, wherein the serum contains an antibody against the diphtheria toxin vaccine, and (ii) the serum is contacted with one or more modified diphtheria toxins, in which case the serum against the modified diphtheria toxin The binding of the antibody forms a complex, and (iii) detects the presence or absence of the complex, where the complex diphtheria toxin detects at least one B cell epitope if the complex is detected. If not lost and a decrease in the amount of complex is detected, the modified diphtheria toxin will have at least one B
Provided herein is a method comprising selecting a modified diphtheria toxin that has lost a cellular epitope and (iv) has lost at least one B cell epitope.

Provided herein are pharmaceutical compositions containing the modified toxin and a pharmaceutically acceptable carrier or excipient.

Provided herein is a method of treating a malignant disease in a mammal and a non-malignant disease, such as GVHD, comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition described herein.

The malignant disease can be a hematological cancer. The malignant disease can be a solid tumor.
The malignant disease can also be metastasis. Typical hematological cancers include, but are not limited to, acute myeloid leukemia, cutaneous T cell lymphoma, relapsed / refractory T cell non-Hodgkin lymphoma, relapsed / refractory B cell non-Hodgkin lymphoma, subcutaneous adipose tissue-like T cell lymphoma, extranodal natural killer / T cell lymphoma nasal type, chronic lymphocytic leukemia, solid tumor and human T cell lymphotropic virus type 1 associated acute T cell leukemia / lymphoma. Representative solid tumors include, but are not limited to, skin, melanoma, lung, pancreas, breast, ovary, colon,
Rectum, stomach, thyroid, larynx, prostate, colorectal, head, neck, eye, mouth, organ, esophagus, chest, bone,
Solid tumors of tissues or organs selected from testis, lymph, bone marrow, bone, sarcoma, kidney, sweat gland, liver, kidney, brain, gastrointestinal tract, nasopharynx, urogenital tract, muscle and other tissues . Metastasis includes, but is not limited to, metastatic tumors of any of the described solid tumors.

  Non-malignant diseases include, for example, GVHD, aGVHD and psoriasis.

Provided herein are methods for enhancing the activity of anti-cancer agents (eg, RNA-transduced DCs, anti-CLTA4 antibodies, MISIIR scFvs, etc.) by administering the DT variant-IL2 fusion proteins described herein. In one embodiment, the DT variant-IL2 fusion protein is administered followed by the anticancer agent. In one non-limiting example, the DT variant-IL2 fusion protein is administered at least 4 days before the anticancer agent.

In addition, anticancer agents (eg, RNA-introduced DC, anti-CLTA4 antibody, MISIIR scF
Provided herein is a method of treating metastatic cancer by reducing or eliminating Tregs by administering vs, etc.) and a DT variant-IL2 fusion protein described herein. Examples of metastatic tumors include metastatic renal cell carcinoma, metastatic prostate cancer, metastatic ovarian cancer, and metastatic lung cancer. In one embodiment, a DT variant-IL2 fusion protein is administered followed by an anticancer agent. In one non-limiting example, D
The T variant-IL2 fusion protein is administered at least 4 days before the anticancer agent.

In another embodiment, an anti-cancer agent (eg, RNA-introduced DC, anti-CLTA4 antibody, MISI
IR scFvs, etc.) and a DT variant-IL2 fusion protein described herein is a method of treating prostate, ovarian, lung or melanoma by reducing or eliminating Tregs by administering a DT variant-IL2 fusion protein described herein. Provided. In one embodiment, DT
A variant-IL2 fusion protein is administered followed by an anticancer agent. In one non-limiting example, the DT variant-IL2 fusion protein is at least 4 of the anticancer agent.
Will be given the day before.

Shows the frequency of allotypes in the donor population. A comparison of the frequency of donor allotypes expressed in the test and social populations is shown. 1 represents DT T cell epitope map and donor response. Figure 3 shows a CD4 + T cell epitope map of DT-1 using overlapping peptides tested against 51 healthy donors. The background response rate (5.6%) is indicated by a red dotted line. Peptides that induce a response that exceeds this threshold contain T cell epitopes (indicated by the a + symbol). Represents DT T cell epitope 1. Epitope 1 was identified using EpiScreen ™ T cell epitope mapping. Three donors reacted to peptide 2 (donors 30, 36 and 47). The proposed 9-mer binding register for peptide 2 (SEQ ID NO: 161) is shown with the indicated p1 and p9 anchor residues. For comparison, peptides 1 and 3 (SEQ ID NOs: 158 and 160) are shown. DT T cell epitope 2 is shown. Epitope 2 was identified using EpiScreen ™ T cell epitope mapping. Four donors reacted to peptide 31 (donors 12, 23, 30 and 36). The proposed 9-mer binding register for peptide 31 (SEQ ID NO: 162) is shown with the indicated p1 and p9 anchor residues. For comparison, peptide 32 (SEQ ID NO: 163) is shown. DT T cell epitope 3 is shown. Epitope 3 was identified using EpiScreen ™ T cell epitope mapping. Three donors reacted to peptide 35 (donors 1, 2 and 35). The proposed 9-mer binding register for peptide 35 (SEQ ID NO: 166) is shown with the indicated p1 and p9 anchor residues. For comparison, peptide 34 (SEQ ID NO: 165) is shown. DT T cell epitope 4 is shown. Epitope 4 was identified using EpiScreen ™ T cell epitope mapping. Three donors reacted to peptide 39 (donors 5, 15 and 50). The proposed 9-mer binding register for peptide 39 (SEQ ID NO: 169) is shown with the indicated p1 and p9 anchor residues. DT T cell epitope 5 is shown. Epitope 5 was identified using EpiScreen ™ T cell epitope mapping. Six donors reacted to peptides 40, 41 and 42. The proposed 9-mer binding register for this region (SEQ ID NO: 173) is shown with the indicated p1 and p9 anchor residues. DT T cell epitope 6 is shown. Epitope 6 was identified using EpiScreen ™ T cell epitope mapping. Three donors (donors 30, 39 and 49) reacted to peptide 49. The proposed 9-mer binding register for this region (SEQ ID NO: 175) is shown with the indicated p1 and p9 anchor residues. DT T cell epitope 7 is shown. Epitope 7 was identified using EpiScreen ™ T cell epitope mapping. Four donors (donors 1, 15, 30, and 35) reacted to peptide 100. The proposed 9-mer binding register for peptide 100 (SEQ ID NO: 178) is shown with the indicated p1 and p9 anchor residues. For comparison, peptide 99 (SEQ ID NO: 176) is shown. A DT T cell epitope map is shown. The position of the CD4 + T cell epitope within the sequence of DT-1 is indicated. T cell epitopes identified by T cell epitope mapping are shown as bars with responding donor identifiers above the sequence. The frequency of the donor allotype response to DT peptide is shown. Figure 5 shows a pie chart representation of the frequency of responding donor allotypes (expressed as%) compared to the frequency expressed in the test population. The analysis was limited to those epitopes that elicited responses greater than 8% of the test population and allotypes expressed with a frequency greater than 5% in the study population. DT peptide stimulation index for unadjusted donor data. A stimulation index highlighted in bold indicates a positive response (SI> 2.0, p <0.05). Numbers highlighted in italics indicate borderline results (SI> 1.9, p <0.05). Donors 13, 20, and 38 were excluded from the analysis because of very low CPM. DT peptide stimulation index for adjusted donor data is shown. A stimulation index highlighted in bold indicates a positive response (SI> 2.0, p <0.05). Numbers highlighted in italics indicate borderline results (SI> 1.9, p <0.05). Donors 13, 20, and 38 were excluded from the analysis because of very low CPM. Wild-type ΔR DT (indicated by diamonds) indicates that transcription / translation of the T7-luc plasmid was inhibited to a greater extent than the null structure (indicated by asterisks) in the IVTT assay. The DT specific antibody indicates that DT bound to the surface of the endothelial cells was detected. FIG. 6 shows binding of DT variants to HUVEC cells (DT-Glu52, CRM mutant) and detection with antibodies using FACS analysis. The black bar graph is the result of using no binder. The hatched hatched bar graph shows assay controls using the various conditions described. The cross-hatched bar graph shows the DT variant in the presence of detection antibody (DT + goat pAb anti-DT (Serotec) + anti-gt-PE). Figure 2 shows detection of antibodies with FACS on binding of ONTAK-A1488 and DT-Glu52-A1488 to HUVEC cells. ONTAK®-A1488 6: 1 D: P (yellow top line, indicated by diamonds); DT-Glu52-A1488 3: 1 D: P (red middle line, indicated by circles); and DT Glu52-A1488 2: 1 D: P (green lower line, indicated by triangles). HUVEC cells were tested for IL-2R expression by FACS; showing that ONTAK®-A1488 was confirmed not to bind via the IL-2 receptor. 2 illustrates a cell membrane integrity assay using propidium iodide and FACS to measure loss of cell membrane integrity after a brief incubation with a toxin. ONTAK® (shown with diamonds), DT-Glu52 (shown with squares) and rhIL-2 (shown with triangles). ONTAK (containing 4 VLS motifs) appears to cause greater membrane damage than either DT-Glu52 (3 VLS motifs) or rhIL-2 (1 VLS motif). Cytotoxicity assays are used to confirm the activity of LS-IL2 T cell epitopes and VLS variant reads selected in the IVTT assay. ONTAK® (diamonds), recombinant human IL-2 (rhIL-2, indicated by squares) and control (indicated by triangles) illustrate that ONTAK® is cytotoxic. FIG. 4 illustrates the ADP ribosylation activity of a variant compared to wild type (WT). The assay threshold was 0.5. Bars marked with “ ^! ” Or “ ^* ” indicate statistically significant results. Illustrates the design of a representative number of epitope variants. Figure 3 illustrates the ADP ribosylation activity of a variant compared to wild type (WT). The assay threshold was 0.5. In vitro transcription / translation inhibition of the target T7-luciferase plasmid by wild type and epitope (Ep) variants of DT was measured using a T7-linked reticulocyte lysate kit and SteadyGlo chemiluminescent reagent (Promega). IC ₅₀ was measured, and the value of wild type DT was divided by the value of each epitope variant to calculate the plotted activity ratio (Y axis). Average values from repeated experiments (n = 3) are shown. 1 = WT activity. 0.5 = threshold for minimum acceptable activity. 2 shows the relative activity of an epitope variant of DT382 compared to wild-type DT382 in inhibiting protein synthesis. 2 shows the relative activity of VLS DT variants compared to wild-type DT in inhibiting protein synthesis. Figure 3 shows the binding of labeled VLS variants to HUVEC cells. DT389-IL2 is shown as a black diamond mark (♦), DT382 is shown as a black square mark (■), DT382 (V7N V29T S292A) is shown as a black triangle mark (▲), and DT382. (V7N V29T I290N) is shown as “x” and BSA is shown as a black circle (●). IC ₅₀ data for modified DT variants compared to wild type and null DT variants is shown. WT is shown as a black diamond (♦), a null DT variant is shown as a white square (□), DT29N is shown as a symbol “+”, and S30G is a black triangle (▲) ) And V148A are shown as asterisks (*). The following T cell map of DAB398 (DT) was generated using iTope ™ T cell epitope prediction software. Regions that may contain T cell epitopes are underlined with possible p1 anchor residues in bold. The location of T cell epitopes identified using in vitro T cell assays is highlighted in box. IC ₅₀ data is shown for the quadruple T cell epitope DT variant. FIG. 5 shows detection of antibodies with conjugated-FACS of ONTAK-488, control DT (ΔR) -488 and BSA-488 against HUVEC cells. ONTAK®-488 2.8: 1 D: P (top line, further indicated by “+”), control DT (ΔR) -488 3: 1 D: P (second line above, BSA-488 7: 1 D: P (middle line, indicated by an asterisk “*”), BSA-488 5: 1 D: P (second line from the bottom, “ X ") and BSA-488 2.6: 1 D: P (bottom line, indicated by triangles" ▲ "). The amino acid sequences of wild type DT382, DT382 variant and null structure DT382 (G53E) are shown. Underlined sequences are vector / tag sequences, enterokinase cleavage sites are highlighted in italics and mutations derived from WT sequences are in bold.

Incorporation of documents Are all publications and patent application specifications mentioned in this specification indicated that each individual publication or patent application specification is incorporated by reference specifically and independently? Are incorporated herein by reference to the same extent.

It is to be understood that this application is not limited to specific formulations or process parameters. Because these can of course be changed. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should also be understood that a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

In accordance with this application, conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art may be used. Such techniques are explained fully in the literature. For example, Sambr
ok et al. , “Molecular Cloning: A Laborato
ry Manual "(1989);" Current Protocols in M "
molecular biology "Volumes I-III [Ausubel,
R. M.M. , Ed. (1994)]; “Cell Biology: A Laborato
ry Handbook "Volumes I-III [J. E. Celis, ed.
(1994)]; “Current Protocols in Immunology.
Volumes I-III [Coligan, J. et al. E. , Ed. (1994)],
“Oligonucleotide Synthesis” (MJ Gait ed.
1984); “Nucleic Acid Hybridization” [B. D. H
ames & S. J. et al. Higgms eds. (1985)]; “Transscript”
"tion and Translation" [B. D. Hames & S. J. et al. Hi
ggins, eds. (1984)]; “Animal Cell Culture” [
R. I. Freshney, ed. (1986)], “Immobilized Cel.
ls And Enzymes "[IRL Press, (1986)]; Perb
al, “A Practical Guide To Molecular Cloni.
ng "(1984). Each of these is hereby incorporated by reference in its entirety.

I. Methods for identifying T cell epitopes Recently, techniques to develop soluble complexes of recombinant MHC molecules in combination with synthetic peptides have begun to be used [Kern, F. et al. et al. , (1998) Nature Media
cine 4: 975-978; Kwok, W .; W. et al. , (2001) Tr
ends in Immunol. 22: 583-588]. These reagents and procedures can bind T-cell clones from peripheral blood samples from human or laboratory animal subjects that can bind specific MHC peptide complexes and are not compatible with screening multiplicity epitopes against a wide variety of MHC allotypes Used to identify presence.

Biological assays of T cell activation remain the most practical option for providing a reading of the ability of a test peptide / protein sequence to elicit an immune response. An example of this type of approach is the study of Petra et al., Which uses a T cell proliferation assay for the bacterial protein staphylokinase and then epitope mapping using a synthetic peptide to stimulate T cell lines [Petra. A. M.M. et al. , (2002
) J. et al. Immunol. 168: 155-161]. Similarly, T cell proliferation assays using synthetic peptides of tetanus toxin protein have resulted in the definition of the immunodominant epitope region of the toxin [Recee J. et al. C. et al. (1993) J. Am. Immunol
. 151: 6175-6184]. WO 99/53038 uses an isolated subset of human immune cells to promote their differentiation in vitro, and culturing and culturing cells in the presence of a synthetic peptide of interest An approach is disclosed in which T cell epitopes in a test protein can be determined by measuring the induced proliferation of T cells.
The same technique has also been described by Stickler et al. [Stickler, M. et al. M
. et al. (2000) J. Am. Immunotherapy 23: 654-660
In both cases, this method is applied to the detection of T cell epitopes within the bacterial subtilisin. Such a technique involves the desired immune cell subset (dendritic cells, CD4 + and / or
Or CD8 + T cells) requires careful application of cell isolation methods and cell culture using multiple cytokine supplements.

International Publication No. WO 02/069232 describes a computational method for defining MHC class II ligands for a number of proteins of therapeutic interest. However, for reasons such as the need for proteolytic processing and other physiological steps that lead to the presentation of immunogenic peptides in vivo, a relatively small subset of the total repertoire of peptides that can be defined by computer-based schemes is ultimately It is clear that it has biological relevance. Thus, in vitro human T cell activation assays can be used to identify regions within the protein sequence of toxins that can support T cell activation, thereby most addressing immunogenicity problems in this protein. Biologically relevant. As used herein, a “T cell epitope” can bind to MHC class II, can stimulate T cells, and / or can be complexed with MHC class II to bind T cells. It refers to an amino acid sequence (not necessarily activated to be measurable).

In accordance with the methods disclosed herein, a synthetic peptide is tested for its ability to elicit a proliferative response in human T cells cultured in vitro. T cells are present in a peripheral blood mononuclear cell (PBMC) layer that can be easily obtained from whole blood samples by well-known means. In addition, PB
MC specimens contain a physiologic ratio of T cells and antigen presenting cells and are therefore a good source of material for surrogate immune responses in vitro. In the operation of such an assay, a stimulation index approximately equal to or greater than 2.0 is a useful measure of induced proliferation. However, the stimulation index is
It may vary depending on the toxin and can be determined by reference to the baseline for each toxin and the corresponding peptide library. In one example of such a test, the stimulation index (SI) is a proliferation score measured against the test peptide (eg, counts per minute of radioactivity when using, for example, ³ H-thymidine incorporation). Number) may be routinely obtained by dividing by the score measured in cells not in contact with the test peptide. Peptides that do not elicit a response may exhibit SI = 1.0, but SI values within the range of 0.8-1.2 may also not be unusual. A number of technical methods can be incorporated into the operation of such assays to ensure confidence in the recorded scores. Typically, all measurements are made in at least 3 iterations and an average score can be calculated. If the calculated SI => 2.0, the individual scores of the three iterations can be examined for evidence of abnormal data. The test peptide is contacted with the cell at at least two concentrations,
The concentration will typically span at least a two-fold concentration difference. Such a concentration range provides an offset to the kinetic dimension for the assay and may be useful when performed at a single time point measurement, eg, plus 7 days. In some assays,
Multiple time course measurements may be performed, and these may also be performed using peptide immunogens provided in at least two concentrations. Similarly, the inclusion of a control peptide, where the majority of PBMC donor samples are expected to be reactive, can be included in each assay plate. Influenza hemagglutinin peptide 307-309, sequence PKYVKQNTLKLA (SEQ ID NO:
201); and the Chlamydia HSP60 peptide sequence KVVDQIKIKSKPVQH (SEQ ID NO: 202) is an example of using a control peptide in such an assay. Alternatively, or in addition, the assay can also use possible total protein antigens, such as hemocyanin from keyhole limpet, against which all PBMC samples are expected to exhibit SI significantly greater than 2.0. I will. Other control antigens for such use will be well known in the art.

The methods disclosed herein can provide an epitope map of a toxin when the map is related to a wide range of possible MHC allotypes. The map will allow the design or selection of modified proteins that may exclude or at least improve the ability of the protein to elicit a T cell driven immune response for the majority of patients who may be administered the protein. It can be well represented. An improvement is a reduction in immune response (ie, reduced immunogenicity) compared to an unmodified protein (eg, more or less about 1.5 times, more or less about 2 times, more or less about 5 times, more or less about 10). Times, more or less about 20 times, more or less about 50 times, more or less about 100 times, more or less about 200 times, more or less about 500 times,
Or this range of these). Alternatively, a protein or toxin with reduced immunogenicity is a% decrease in its ability to elicit an immune response compared to an unmodified protein (eg, less than about 1%, less than about 2%, less than about 3%, about 4%). %, Less than about 5%, less than about 10%, less than about 20%, less than about 50%, less than about 100%, and ranges thereof. Therefore, in the performance of the screening method, PBMC-derived T cells from an untreated donor are
Over at least 90% of the existing MHC class II repertoire (HLA-D) in the human population
R) is collected from a pool of donors of sufficient immunodiversity to provide samples. If naive T cell responses are to be detected against a given synthetic peptide, the peptide actually contacts PBMC specimens derived from multiple donors in isolation. The number of donors (or “donor pool” size) does not appear to be practically less than 20 unrelated individuals and all samples in the donor pool may be pre-selected according to their MHC class II haplotype .

As used herein, the term “naïve donor” refers to a subject that has not been previously exposed to toxins environmentally, by vaccination, or by other means such as, for example, blood transfusion.

It is noted that people in certain countries are regularly vaccinated against toxins or exposed to environmental sources of exogenous toxins such as diphtheria toxin and toxin-like proteins. In such people, the possibility of recall response exists as a measure by the increased SI score.

When screening for T cell epitopes, T cells can be obtained from peripheral blood samples from a number of different healthy donors, but from healthy donors that have not therapeutically received proteins. If necessary, a patient's blood sample can be tested for the presence of a particular polypeptide using a conventional assay such as an ELISA that uses antibodies to identify the presence or absence of one or more polypeptides. it can. The assay is performed using PBMCs cultured in vitro using conventional methods known in the art and contacting the PBMC with a synthetic peptide species (ie, library) representing the protein of interest. And measurement of peptide-induced T cell activation such as incubation and cell proliferation for an appropriate period of time. The measurement can be any suitable means and may be performed using, for example, uptake of H ³ -thymidine, so that accumulation of H ^{3 on} the cellular material is facilitated using experimental equipment. Measured. The degree of cell proliferation for each combination of PBMC sample and synthetic peptide can be examined relative to the degree of cell proliferation observed in non-peptide treated PBMC samples. Reference can also be made to the proliferative response observed after treatment with one or more peptides with the expected proliferative effect. In this regard, it is advantageous to use peptides with a wide range of known MHC restrictions, in particular peptide epitopes with MHC restrictions on DP or DQ isoforms, but the invention is not limited to the use of such restriction peptides . Such peptides are described above for example with respect to influenza hemagglutinin and Chlamydia HSP60.

In one non-limiting example, a T cell epitope for diphtheria toxin (DT) is mapped and subsequently modified using the methods described herein. To facilitate the assembly of epitope maps for DT, a library of synthetic peptides is created. Each of the peptides is 15 amino acid residues in length, and each overlaps a series of subsequent peptides by 12 amino acid residues. That is, each series of peptides incrementally added three more amino acids to the analysis. In this way, a given contiguous pair of peptides mapped 18 amino acids in a contiguous sequence. One method for defining a T cell map for DT using a naive T cell assay is illustrated in Example 7. Each of the peptides identified by the method of defining the T cell map of the toxin is MH
It suggests that C class II can bind and be involved in at least one cognate TCR with sufficient affinity to induce a proliferation explosion that can be detected in the assay system.

II. Methods for Modifying Toxins The toxin molecules described herein can be prepared in any of several ways, including the use of recombinant methods. The protein sequences and information provided herein can be used to infer a polynucleotide (DNA) encoding an amino acid sequence. This is, for example, DNSst
ar software suite [DNAstar Inc, Madison, Wi
s. , USA] or the like. Such a polynucleotide encoding a polypeptide, or important homologues, variants, truncations, extensions or other modifications thereof are contemplated herein.

Provided herein are methods for mapping (identifying) a T cell epitope of a toxin and modifying the epitope such that the modified sequence inhibits (partially or completely) the induction of a helper T response. Is done. Modifications include amino acid substitutions, deletions, or insertions made at the codons of the polynucleotides that encode the modified polypeptides to affect similar changes. Codons encoding amino acid residues are well known in the art. Recombinant DNA methods can be used to achieve directed mutagenesis of the target sequence, and many such methods are available, as described herein and known in the art as described above. Yes. In general, the technique of site-directed mutagenesis is well known. Briefly, a bacteriophage vector is used that produces a single-stranded template for oligonucleotide-directed PCR mutagenesis. Phage vectors (eg, M13) are commercially available and their use is generally well known in the art. Similarly, double-stranded plasmids are also commonly used in site-directed mutagenesis that eliminates the step of transferring the polynucleotide of interest from the phage to the plasmid. Synthetic oligonucleotide primers with a given mutated sequence can be used to direct in vitro synthesis of modified (desired mutant) DNA from this template, and heteroduplex DNA can be Used to transform competent E. coli for growth selection and identification. Alternatively, a pair of primers
By annealing to two separate strands of a double stranded vector, both corresponding complementary strands with the desired mutation in the PCR reaction can be synthesized simultaneously.

In one embodiment, Quick Change site-directed mutagenesis using a plasmid DNA template as reported by Sugimoto et al. Can be used (Sugimoto et al., Anal. Biochem., 179 (2):
309-311 (1989)). P of the plasmid template containing the insertion target gene of the insert
CR amplification is achieved using two synthetic oligonucleotide primers containing the desired mutation. Oligonucleotide primers, each complementary to the opposite strand of the vector, are extended during temperature cycling by mutagenic grade PfuTurbo DNA polymerase. Upon incorporation of the oligonucleotide primer, a mutated plasmid containing a twisted nick is generated. The amplified unmethylated product is treated with DpnI to digest the methylated parent DNA template and select newly synthesized DNA containing the mutation. Because DNA isolated from most E. coli strains is dam methylated, it is susceptible to DpnI digestion specific for methylated and hemimethylated DNA. The reaction product is transformed into a high efficiency strain of E. coli to obtain a plasmid with the desired modification. Alternative methods of inducing amino acid modifications in polypeptides are well known in the art and can be used herein.

Suitable modifications for proteins may include amino acid substitutions of specific residues or combinations of residues. For elimination of T cell epitopes, amino acid substitutions are made at appropriate positions or amino acid residues within the amino acid sequence that are predicted to achieve suppression or elimination of the activity of the T cell epitope. Indeed, it will be preferred that the appropriate position or amino acid residue is the same as the amino acid residue that binds in one of the pockets provided within the MHC class II binding groove.
Such modifications can alter the binding within the first pocket of the cleft at the so-called “P1” or “P1 anchor” position of the peptide. It is recognized that the nature of the binding interaction between the P1 anchor residue of the peptide and the first pocket of the MHC class II binding groove is a major determinant of overall binding affinity for all peptides. Appropriate substitutions at this position in the amino acid sequence will generally incorporate amino acid residues that are difficult to adapt within a pocket (eg, substitutions for more hydrophilic residues). Amino acid residues of the peptide at the same position as bonds in other pocket regions within the MHC binding cleft are also considered and fall within this range.

It is understood that a single amino acid modification within a given possible T cell epitope represents a pathway that can eliminate one or more T cell epitopes. Combinations of modifications within a single epitope can be considered and may be appropriate when independently defined epitopes overlap each other. Also, amino acid modifications (alone within a given epitope or in combination within a single epitope) are positions that differ from “pocket residues” with respect to the MHC class II binding groove, but at any position within the amino acid sequence. It may be done. Modifications may be made with reference to homologous or structural methods that are known in the art and that occur using known computer methods described herein and are known structurally known to polypeptides. It may be based on features. The change (modification) can be intended to restore the structure or biological activity of the variant molecule. Such compensatory changes and changes may also include deletion or addition (insertion) of specific amino acid residues from the polypeptide. Also, modifications can be made that alter the structure and / or suppress the biological activity of the molecule and eliminate T cell epitopes and thus reduce the immunogenicity of the molecule. Any form of modification is contemplated herein.

Another means of removing epitopes from protein molecules is described in WO 02/0692.
Co-operation of a naïve T cell activation assay scheme as outlined herein with computer tools developed according to the scheme described in specification 32, which is also fully incorporated herein by reference. Is use. The software simulates the process of antigen presentation at the level of polypeptide MHC class II binding interaction to obtain a binding score for a given polypeptide sequence. Such scores are measured for many of the major MHC class II allotypes present in the population. Since this scheme can test any polypeptide sequence, the consequences of amino acid substitutions, additions or deletions with respect to the ability of the polypeptide to interact with the MHC class II binding groove can be predicted. Thus, new sequence compositions can be designed that contain a reduced number of amino acids that can interact with MHC class II, thereby functioning as immunogenic T cell epitopes. If a biological assay using one given donor sample can assess binding to up to 4 DR allotypes, the computational method can test the same polypeptide sequence using> 40 allotypes simultaneously. In practice, this approach can direct the design of new sequence variants that are altered in their ability to interact with multiple MHC allotypes. As will be apparent to those skilled in the art, numerous alternative sets of substitutions can be reached that achieve the goal of removing unwanted epitopes. However, the resulting sequences are recognized as being closely homologous to the specific compositions disclosed herein and are therefore within the scope of this application.

A computer for the identification of MHC class II ligands and for the design of sequence analogs lacking MHC class II ligands in cooperation with epitope mapping and optionally a retest using a biology-based assay of T cell activation A combined approach using tools is another method and embodiment of the present application. The general method of this embodiment consists of the following steps:
i) identifying an epitope region capable of activating T cells using a naive T cell activation assay and a synthetic peptide that collectively encompasses the protein sequence of interest;
ii) Analyzing the epitope region identified in step (i) using a computational scheme that simulates the binding of a peptide ligand to one or more MHC allotypes, thereby making the MHC class within the epitope region Identifying a II ligand;
iii) no longer bind MHC class II or bind with a lower affinity to a smaller number of MHC allotypes using a computational scheme that simulates the binding of peptide ligands to one or more MHC allotypes Identifying sequence analogues of MHC ligands contained within the epitope region to be treated, and optionally iv) encompassing the identified epitope region within the protein of interest or in collection, naive T cell activity Using a naive T cell activation assay and a synthetic peptide to test the sequence analogs in the activation assay simultaneously with the wild type (parent) sequence;
Consists of.

In one embodiment, a method of preparing a modified toxin that exhibits reduced immunogenicity compared to an unmodified toxin identifies at least one T cell epitope within the amino acid sequence of the toxin and at least one identified Modification of at least one amino acid residue within the T cell epitope.

In another embodiment, a modified toxin that exhibits reduced immunogenicity compared to an unmodified toxin identifies at least one T cell epitope within the amino acid sequence of the toxin, and at least one identified T cell epitope. Produced by a method that modifies at least one amino acid residue.

In yet another embodiment, the method of selecting a modified toxin that exhibits reduced immunogenicity compared to an unmodified toxin identifies at least one T cell epitope within the amino acid sequence of the toxin;
At least one amino acid residue within at least one identified T cell epitope is modified and a modified toxin is selected that exhibits reduced immunogenicity compared to the unmodified toxin.
DT T cell epitopes Also provided herein are DT variants having at least one modification in one or more T cell epitopes. Seven T cell epitopes were identified in DT by the methods described herein and further by the method described in Example 9. The seven T cell epitopes contain diphtheria toxin amino acid sequences as shown in SEQ ID NOs: 181, 184, 187, 190, 193, 196 and 199. SEQ ID NO: 1 as described herein
81 corresponds to amino acid residues 1-27 of SEQ ID NO: 2 or 200, SEQ ID NO: 184 corresponds to amino acid residues 85-117 of SEQ ID NO: 2, and SEQ ID NO: 187 is SEQ ID NO:
Corresponding to 2 or 200 amino acid residues 95-127, SEQ ID NO: 190 is SEQ ID NO: 2
Or corresponding to 200 amino acid residues 104-136, SEQ ID NO: 193 is SEQ ID NO: 2
Or corresponding to 200 amino acid residues 112-144, SEQ ID NO: 196 is SEQ ID NO: 2
Or corresponding to 200 amino acid residues 136-168 and SEQ ID NO: 199 is SEQ ID NO:
It corresponds to 2 or 200 amino acid residues 286-318. These epitopes further contain the epitope (9-mer binding register) and the core 9-mer amino acid sequence, which is the most preferred binding register for MHC class II binding of adjacent amino acid residues. Although the core 9-mer binding register is thought to be the primary epitope, residues located outside the 9-mer binding register interact with MHC class II molecules and stabilize the peptide / MHC class II complex. It has been shown to support sex. The seven identified DT T cell epitope core 9-mer binding registers are SEQ ID NOs: 161, 164, 167, 169,
It has the amino acid sequence of 173, 175 and 178.

The T cell epitopes described herein can be further characterized by the region of the epitope. Such a region contains the epitope core, N-terminus and C-terminus. As used herein, “epitope core” refers to the core 9-mer amino acid sequence of a T cell epitope. The epitope core can further contain 0, 1, 2, or 3 amino acid residues adjacent to the N-terminal and / or C-terminal core 9-mer amino acid sequence. Thus, the epitope core can range in length from about 9 amino acids up to about 15 amino acids in certain embodiments.

As used herein, “N-terminus” refers to an amino acid adjacent to the N-terminus of the epitope core, and is adjacent to and upstream of the N-terminus of the epitope core, at least 1, 2, 3, 4, Includes 5, 6, 7, 8, or 9 amino acids.

As used herein, “C-terminus” refers to an amino acid adjacent to the C-terminus of the epitope core, and is adjacent to and downstream of the C-terminus of the epitope core, at least 1, 2, 3, 4, Includes 5, 6, 7, 8, or 9 amino acids.

DT T cell epitope 1 consists of a 9-mer peptide having the amino acid sequence shown as VDSSKSFVM (SEQ ID NO: 161). As described herein, the elimination of T cell epitopes is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 of the epitope core. 0, 1, 2, by modifying 1, 11, 12, 13, 14 or 15 amino acids and / or adjacent to the N-terminus and / or C-terminus of the epitope core
This can be achieved by modifying 3, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues.

In one non-limiting example, D having the amino acid sequence shown as SEQ ID NO: 181
Provided herein are diphtheria toxins having one or more amino acid modifications within the epitope core of TT cell epitope 1, wherein the amino acid modifications are by insertions, deletions or substitutions. . The core epitope of DT T cell epitope 1 is amino acid residues 7-15, 6-16, 5-17 of the amino acid sequence shown in SEQ ID NO: 181,
4-18 or any combination thereof can be included. Also provided herein is a diphtheria toxin having one or more amino acid modifications at the N-terminus and / or C-terminus of DT T cell epitope 1 having the amino acid sequence set forth in SEQ ID NO: 181. The N-terminus of DT T cell epitope 1 contains amino acid residues 1-6, 1-5, 1-4, 1-3 of the amino acid sequence shown in SEQ ID NO: 181 or any combination thereof.
The C-terminus of DT T cell epitope 1 contains amino acid residues 16-24, 17-25, 18-26, 19-27 of the amino acid sequence set forth in SEQ ID NO: 181 or any combination thereof. Also provided herein are modified diphtheria toxins having one or more amino acid modifications in the epitope core of SEQ ID NO: 181 and within the N-terminus and / or C-terminus of SEQ ID NO: 181. In one non-limiting example, the epitope core P1
The valine residue can be replaced with any amino acid residue to modify and / or eliminate the identified T cell epitope. In one embodiment, the P1 valine residue of the epitope core is a surface with a polar portion of the residue exposed on the DT molecule and hydrophobic to modify and / or eliminate the identified T cell epitope Polar amino acid residues can be substituted so that the region is embedded. Examples of polar amino acid residues include, but are not limited to, histidine (H), glycine (G), lysine (K), serine (S), threonine (T
), Cysteine (C), tyrosine (Y), asparagine (N), aspartic acid / aspartate (D), glutamic acid / glutamate (E) and glutamine (Q). It will be appreciated that these modifications are applicable to the other epitopes described below.

DT T cell epitope 2 consists of a 9-mer peptide having the amino acid sequence shown as VDNAETIKK (SEQ ID NO: 164). This elimination of T cell epitopes can be achieved by modifying 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of the epitope core and / or Or 0, 1, 2, 3, N-terminal and / or C-terminal,
This can be accomplished by modifying 4, 5, 6, 7, 8, 9, or more amino acid residues. In one non-limiting example, a diphtheria toxin having one or more amino acid modifications within the epitope core of SEQ ID NO: 184, wherein the amino acid modification is by insertion, deletion or substitution, Provided in the specification. DT
The core epitope of T cell epitope 2 contains amino acid residues 13-21, 12-22, 11-23, 10-24 of the amino acid sequence shown as SEQ ID NO: 184, or any combination thereof. Also provided herein is a diphtheria toxin having one or more amino acid modifications within the N-terminus and / or C-terminus of DT T cell epitope 2 having the amino acid sequence set forth in SEQ ID NO: 184. The N-terminus of DT T cell epitope 2 is amino acid residues 1-9, 2-10, 3-11, 4 of the amino acid sequence shown as SEQ ID NO: 184
Contains -12 or any combination thereof. The C-terminus of DT T cell epitope 2 is amino acid residues 22-30, 23-31 of the amino acid sequence shown as SEQ ID NO: 184.
, 24-32, 25-33, or any combination thereof. Also provided herein are modified diphtheria toxins having one or more amino acid modifications in the epitope core of SEQ ID NO: 184 and within the N-terminus and / or C-terminus. In one non-limiting example, the P1 valine residue of the core 9-mer can be replaced with any amino acid residue to modify and / or eliminate the identified T cell epitope.

DT T cell epitope 3 consists of a 9-mer peptide having the amino acid sequence shown as LGLSLTEPL (SEQ ID NO: 167). This elimination of T cell epitopes can be achieved by modifying 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of the epitope core and / or Or 0, 1, 2, 3, N-terminal and / or C-terminal,
This can be accomplished by modifying 4, 5, 6, 7, 8, 9, or more amino acid residues. In one non-limiting example, a diphtheria toxin having one or more amino acid modifications within the epitope core of SEQ ID NO: 187, wherein the amino acid modification is by insertion, deletion or substitution, Provided in the specification. The core epitope of DT T cell epitope 3 can contain amino acid residues 13-21, 12-22, 11-23, 10-24 of the amino acid sequence shown as SEQ ID NO: 187, or any combination therein. . Also provided herein is a diphtheria toxin having one or more amino acid modifications within the N-terminus and / or C-terminus of DT T cell epitope 3 having the amino acid sequence set forth in SEQ ID NO: 187. The N-terminus of DT T cell epitope 3 is amino acid residues 1-9 of the amino acid sequence shown as SEQ ID NO: 187,
2-10, 3-11, 4-12 or any combination thereof. The C-terminus of DT T cell epitope 3 is amino acid residue 22 of the amino acid sequence shown as SEQ ID NO: 187.
-30, 23-31, 24-32, 25-33 or any combination thereof. Also provided herein are modified diphtheria toxins having one or more amino acid modifications in the epitope core of SEQ ID NO: 187 and within the N-terminus and / or C-terminus.
In one non-limiting example, the core 9-mer P1 lysine residue is an exposed surface on the DT molecule with a polar portion of the residue to modify and / or eliminate the identified T cell epitopes and It can be substituted with polar amino acid residues so that the hydrophobic and hydrophobic regions are embedded. In yet another non-limiting example, the P6 threonine and / or P7 glutamate position of the core 9-mer can be replaced with any amino acid to modify and / or eliminate the identified T cell epitope.

DT T cell epitope 4 consists of a 9-mer peptide having the amino acid sequence shown as MEQVGTEEF (SEQ ID NO: 169). This elimination of T cell epitopes can be achieved by modifying 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of the epitope core and / or Or 0, 1, 2, 3, N-terminal and / or C-terminal,
This can be accomplished by modifying 4, 5, 6, 7, 8, 9, or more amino acid residues. In one non-limiting example, a diphtheria toxin having one or more amino acid modifications within the epitope core of SEQ ID NO: 190, wherein the amino acid modification is by insertion, deletion or substitution, Provided in the specification. The core epitope of DT T cell epitope 4 can contain amino acid residues 13-21, 12-22, 11-23, 10-24 of the amino acid sequence shown as SEQ ID NO: 190, or any combination thereof. . Also provided herein is a diphtheria toxin having one or more amino acid modifications within the N-terminus and / or C-terminus of DT T cell epitope 4 having the amino acid sequence set forth in SEQ ID NO: 190. The N-terminus of DT T cell epitope 4 is amino acid residues 1-9, 2 of the amino acid sequence shown as SEQ ID NO: 190
Contains -10, 3-11, 4-12 or any combination thereof. The C-terminus of DT T cell epitope 4 is the amino acid residue 22- of the amino acid sequence shown as SEQ ID NO: 190
30, 23-31, 24-32, 25-33 or any combination thereof.
Also provided herein are modified diphtheria toxins having one or more amino acid modifications in the epitope core of SEQ ID NO: 190 and within the N-terminus and / or C-terminus. In one non-limiting example, the P9 threonine and / or P7 glutamic acid residues of the core 9-mer can be replaced with any amino acid to modify and / or eliminate the identified T cell epitope.

DT T cell epitope 5 consists of a 9-mer peptide having the amino acid sequence shown as FIKRFGDGA (SEQ ID NO: 173). This elimination of T cell epitopes can be achieved by modifying 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of the epitope core and / or Or 0, 1, 2, 3, N-terminal and / or C-terminal,
This can be accomplished by modifying 4, 5, 6, 7, 8, 9, or more amino acid residues. In one non-limiting example, a diphtheria toxin having one or more amino acid modifications within the epitope core of SEQ ID NO: 193, wherein the amino acid modification is by insertion, deletion or substitution, Provided in the specification. The core epitope of DT T cell epitope 5 can contain amino acid residues 13-21, 12-22, 11-23, 10-24 of the amino acid sequence shown as SEQ ID NO: 193, or any combination thereof. . Also provided herein is a diphtheria toxin having one or more amino acid modifications within the N-terminus and / or C-terminus of DT T cell epitope 5 having the amino acid sequence set forth in SEQ ID NO: 193. The N-terminus of DT T cell epitope 5 is amino acid residues 1-9, 2 of the amino acid sequence shown as SEQ ID NO: 193.
Contains -10, 3-11, 4-12 or any combination thereof. The C-terminus of DT T cell epitope 5 is the amino acid residue 22- of the amino acid sequence shown as SEQ ID NO: 193.
30, 23-31, 24-32, 25-33 or any combination thereof.
Also provided herein are modified diphtheria toxins having one or more amino acid modifications in the epitope core of SEQ ID NO: 193 and within the N-terminus and / or C-terminus. In one non-limiting example, the core nonamer P4 arginine residue, P6 glycine residue, P
7 aspartic acid residues and / or P9 alanine residues, or combinations thereof,
Identified T cell epitopes can be substituted to modify and / or eliminate.

DT T cell epitope 6 consists of a 9-mer peptide having the amino acid sequence shown as VEYINNWEQ (SEQ ID NO: 175). This elimination of T cell epitopes can be achieved by modifying 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of the epitope core and / or Or 0, 1, 2, 3, N-terminal and / or C-terminal,
This can be accomplished by modifying 4, 5, 6, 7, 8, 9, or more amino acid residues. In one non-limiting example, a diphtheria toxin having one or more amino acid modifications within the epitope core of SEQ ID NO: 196, wherein the amino acid modification is by insertion, deletion or substitution, Provided in the specification. The core epitope of DT T cell epitope 6 can contain amino acid residues 13-21, 12-22, 11-23, 10-24 of the amino acid sequence shown as SEQ ID NO: 196, or any combination thereof. . Also provided herein is a diphtheria toxin having one or more amino acid modifications within the N-terminus and / or C-terminus of DT T cell epitope 6 having the amino acid sequence set forth in SEQ ID NO: 196. The N-terminus of DT T cell epitope 6 is amino acid residues 1-9, 2 of the amino acid sequence shown as SEQ ID NO: 196.
Contains -10, 3-11, 4-12 or any combination thereof. The C-terminus of DT T cell epitope 5 is amino acid residue 22- of the amino acid sequence shown as SEQ ID NO: 196.
30, 23-31, 24-32, 25-33 or any combination thereof.
Also provided herein are modified diphtheria toxins having one or more amino acid modifications in the epitope core of SEQ ID NO: 196 and within the N-terminus and / or C-terminus. In one non-limiting example, the core 9-mer P1 valine is a surface with an exposed hydrophilic portion and a hydrophobic region embedded in the protein to modify and / or eliminate identified T cell epitopes. Can be substituted with polar amino acid residues.

DT T cell epitope 7 consists of a 9-mer peptide having the amino acid sequence shown as LEKTTAALS (SEQ ID NO: 178). This elimination of T cell epitopes can be achieved by modifying 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of the epitope core and / or Or 0, 1, 2, 3, N-terminal and / or C-terminal,
This can be accomplished by modifying 4, 5, 6, 7, 8, 9, or more amino acid residues. In one non-limiting example, a diphtheria toxin having one or more amino acid modifications within the epitope core of SEQ ID NO: 199, wherein the amino acid modification is by insertion, deletion or substitution, Provided in the specification. The core epitope of DT T cell epitope 7 can contain amino acid residues 13-21, 12-22, 11-23, 10-24 of the amino acid sequence shown as SEQ ID NO: 199, or any combination thereof. . Also provided herein is a diphtheria toxin having one or more amino acid modifications within the N-terminus and / or C-terminus of DT T cell epitope 7 having the amino acid sequence set forth in SEQ ID NO: 199. The N-terminus of DT T cell epitope 7 is amino acid residues 1-9, 2 of the amino acid sequence shown as SEQ ID NO: 199.
Contains -10, 3-11, 4-12 or any combination thereof. The C-terminus of DT T cell epitope 7 is the amino acid residue 22- of the amino acid sequence shown as SEQ ID NO: 199.
30, 23-31, 24-32, 25-33 or any combination thereof.
Also provided herein are modified diphtheria toxins having one or more amino acid modifications in the epitope core of SEQ ID NO: 199 and within the N-terminus and / or C-terminus. In one non-limiting example, the core 9-mer P1 leucine can be replaced with any amino acid to modify and / or eliminate the identified T cell epitope.

In one embodiment, the modified diphtheria toxin is V7S, V7T, D8E, S9A,
S9T, V29S, V29T, V29N, V29D, D30E, S31N, I290T,
D291E, S292A, S292T, V97A, V97T, V97D, L107N, M
116A, M116Q, M116N, F124H, V148A, V148T, L298A
And one or more modifications selected from among L298N.

In one embodiment, the modified diphtheria toxin is V7N, V7T, V29N, V29.
Contains two modifications selected from among T and V29D. Modifications include, but are not limited to, V7N V29N, V7N V29T, V7N V29D, V7T V29N
, V7T V29T or V7T V29D.

In one embodiment, the modified diphtheria toxin is V7D, V7N, V7T, L107.
Contains three modifications selected from A, L107N and F124H. Modifications include, but are not limited to, V7D L107A F124H, V7D L107N F1
24H, V7N L107A F124H, V7N L107N F124H, V7T
L107A F124H and V7T L107N F124H.

In one embodiment, the DT variant has 4 modifications, eg V7D V97D.
L107N F124H, V7N V97D L107N F124H, V7T V97
A L107N F124H, V7T V97D L107N F124H, V7T V
97T L107N F124H, V7D V97A L107N F124H, V7D
V97T L107N F124H, V7N V97A L107N F124H, V7N V97T L107N F124H, etc. are contained.

In addition to the examples and embodiments described above, modified diphtheria toxins having one or more amino acid modifications in one or more T cell epitopes are contemplated herein. In one non-limiting example, provided herein are diphtheria toxins having at least one modification in at least one T cell epitope. In another non-limiting example:
At least one of said 1, 2, 3, 4, 5, 6, or 7 T cell epitopes
Provided herein are diphtheria toxins having one modification. Further non-limiting examples include diphtheria toxins having two or more amino acid modifications at two or more T cell epitopes. Any combination of amino acid modifications within any number of the aforementioned DT T cell epitopes is contemplated herein.

DT T cell epitopes and allotype frequencies Individual epitopes found within an antigen can be preferentially presented by specific MHC class II allotypes, as well as other specific epitopes within the same antigen are MHC class II May not be presented on the molecule. Specific epitopes and specific MCH class I
Such association with I molecules has been shown to depend on individual MHC class II allotypes. The association of specific epitopes with specific allotypes can also be observed when DT is modified for removal of T cell epitopes. Such considerations can allow for very specific modifications of DT molecules for specific allotypes (eg, for a specific population of subjects with certain MHC class II allotypes). The MHC class II allotype of one or more subjects can be readily determined by genotyping methods known in the art, so the association of a DT T cell epitope with a given allotype is a toxin modification adapted to that allotype. To be easily identified. DT
Identification of the association between the T cell epitope and the MHC class II allotype is shown in Example 9, Table 5 and FIG. Herein, M identified for a given epitope of a toxin.
Modified toxins with T cell epitope modifications compatible with HC class II association are contemplated.

Based on the teachings herein, these methods can be applied to the other toxins described herein. For example, the amino acid sequences of toxins that have been analyzed for MHC association and T cell epitopes can be readily used. In addition, toxins that have not been analyzed for MHC association can also be analyzed and tested throughout the specification and using the methods described in the examples below.

Selection of B Cell Epitopes In addition to the identification and modification of T cell epitopes as described herein, identification and modification of B cell epitopes can further reduce the immunogenicity of the toxin. Serological methods, computational methods, or a combination of both methods can be used to identify B cell epitopes within toxins or modified toxins. Serological methods include immunogenic molecules of a subject, such as proteins,
The ability to generate antibodies against peptides and polypeptides is used. If the toxin or modified toxin is one that has been previously administered to the population, for example for vaccination, the antibody response generated in the subject is used to screen for toxins or modified toxins for B cell epitopes. it can. In one non-limiting example, toxins that already have their modified T cell epitopes as disclosed herein (eg, diphtheria toxin) are further screened to identify B cell epitopes. be able to. Peptide libraries based on the sequences of modified toxins can be synthesized and sera from toxin vaccinated donors containing antibodies against the toxin can be tested for the ability to bind to peptides in the peptide library. Various methods and assays such as, but not limited to, ELISA, RIA, and Western blotting are well known in the art, and include one or more peptides that bind to antibodies from sera of toxin vaccinated donors. Can be used to identify. These one or more peptides that bind the antibody in the donor serum present a B cell epitope within the toxin sequence. After identification of the modified toxin B cell epitope, the amino acid residues corresponding to the B cell epitope can be modified and the modified toxin is re-sorted against the donor serum. This process can be performed multiple times to further reduce the immunogenicity of the modified toxin.
As recognized herein for the identification of T cell epitopes, computer methods utilizing known B cell epitope databases and predictive protein modeling programs are used to identify B cell epitopes within toxins or modified toxins. Can do. Once such an epitope has been identified and modified by computer methods, the modified toxin can be screened against vaccinated donor sera as described herein. Computer B
Cell epitope selection methods can be used alone or in combination with peptide library B cell epitope selection for identification and modification of B cell epitopes in toxins.
Optionally, modified toxins can be screened prior to and / or subsequent to B cell epitope selection. Also contemplated herein is the selection of B cell epitope modified toxins for the presence of any T cell epitope that may have occurred during modification of the B cell epitope.

The invention application described herein is a method of selecting a modified toxin that has lost at least one B cell epitope, wherein a serum sample is obtained from at least one subject immunized with the toxin. And the serum contains an antibody against the toxin, the serum is contacted with one or more modified toxins (the binding of the antibody to the modified toxin forms a complex), and the complex (If the complex is detected, the modified toxin has not lost at least one B cell epitope, and if a decrease in the amount of complex is detected, the modified toxin is at least A modified toxin comprising selecting a modified toxin that has lost one B cell epitope) and at least one B cell epitope, said modified toxin comprising at least one B cell epitope It encompasses a method for selecting a losing are). In one non-limiting embodiment, the reduction in the amount of complex is, for example, about 1.5 times less, about 2 times less, about 5 times less, about 10 times the amount observed with unmodified toxin. It can be less, about 20 times less, about 50 times less, about 100 times less, about 200 times less, or about 500 times less.

IV. Vascular Leakage Syndrome Cell damage, particularly endothelial cell damage, may be caused to the patient regardless of whether it is caused by a toxin or a septic shock, such as by being bitten by a snake, or by a therapeutic agent such as an immunotoxin or interleukin. Still a problem.

VLS is often observed during bacterial sepsis and may be accompanied by IL-2 and various other cytokines (Baluna and Vitetta, Immunopharmac
olology, 37: 117-132, 1996). The underlying mechanism of VLS is unclear, and is thought to involve a cascade of events initiated within endothelial cells (EC) and is also thought to involve an inflammatory cascade and cytokines (Engert et al., In
: Clinical Applications of Immunotoxins, F
rankel (ed), 2: 13-33, 1997). VLS is a vascular endothelial cell (EC)
With a complex etiology with fluid and protein spills resulting in damage to the body and interstitial edema, weight gain and its most severe forms of kidney damage, aphasia and pulmonary edema (Sausville
e and Vitetta, In: Monoclonal Antibody-Bas
ed Therapy of Cancer, Grossbard (ed.), 4:81
-89, 1997; Baluna and Vitetta, Immunopharma.
colology, 37: 117-132, 1996; Engert et al. , In:
Clinical Applications of Immunotoxins, Fr
ankel (ed), 2: 13-33, 1997). Vascular leakage syndrome (VLS) has been a serious problem associated with all immunotoxins so far tested in humans, as well as cytokines such as interleukin 2 (IL-2), TNF and adenoviral vectors. (Rosenberg et al., N. Engl. J. Med., 31
6: 889-897, 1987; Rosenstein et al. , J .; Immun
ol. 137: 1735-1742, 1986).

Antibody conjugate peptides derived from ricin toxin A chain containing modified sequences at residues L74, D75, V76 showed a decrease (US Pat. No. 6,566,500 to Vitetta et al.). Thus, (x) one or more amino acid deletions or mutations in the D / E (y) sequence (s) and / or one or more flanking of a toxin, eg, diphtheria toxin It is contemplated that the residues may reduce or suppress the ability of toxin molecules containing these sequences to induce EC damage. Being able to produce one or more polypeptides containing at least one mutation motif and / or one or more flanking residues that reduce or eliminate the EC damaging activity of such chemicals. Be expected.

A composition having reduced VLS promoting ability based on a mutation of (x) D / E (y) or (x) D / E (y) T sequence in a polypeptide, comprising: Compositions that remove or alter each such sequence and methods of using the same are described below. Thus, it will be understood that all methods described herein for producing polypeptides with reduced VLS promoting ability can be applied to produce polypeptides with reduced EC damage activity. All such methods and compositions identified or produced by such methods and equivalents thereof are encompassed by the present invention.

In certain embodiments, the application provides for diseases such as, but not limited to, malignant diseases such as cutaneous T cell lymphoma, relapsed / refractory T cell non-Hodgkin lymphoma, relapsed / refractory B cell non-Hodgkin lymphoma, subcutaneous fat Tissue-like T-cell lymphoma, extranodal natural killer / T-cell lymphoma nasal type, chronic lymphocytic leukemia and human T-cell lymphotropic virus type 1-related acute T-cell leukemia / lymphoma, etc .; non-malignant diseases such as grafts A modified toxin composition for the manufacture of therapeutics for host disease and psoriasis and the like, as well as damage to endothelial cells during the progression of such disease (ie, VLS), compared to the sequence of the unmodified toxin composition At least one of the sequences containing removed or altered (x) D / E (y) and / or (x) D / E (y) T
Use of a modified toxin composition having 1 amino acid is provided.

The reduction or elimination of vascular leakage syndrome (VLS) as a side effect represents a significant advance because it improves the “risk-benefit ratio” of protein therapeutics, particularly protein therapeutics of the immunotoxin and fusion toxin subclasses (Baluna et al. al., Int. J. Immun
pharmacology, 18 (6-7): 355-361 (1996); Balu
na et al. , Immunopharmacology, 37 (No. 2-3):
117-132 (1997), Bascon, Immunopharmacology,
39 (3): 255 (1998)). The ability to develop fusion proteins consisting of cytotoxins and unique targeting domains (cell binding domains in the case of immunotoxins), i.e. single-chain molecules, is essential for autoimmune diseases such as rheumatoid arthritis and psoriasis, graft rejection and other The development of therapeutics for non-malignant medical applications could be facilitated (Chaudary et al.
, Proc. Natl. Acad. Sci. USA, 87 (23): 9491-9494.
(1990); Frankel et al. , In Clinical Applic
ofs of Immunotoxins Scientific Publics
hing Services, Charleston S .; C. , (1997), Kne
chtle et al. , Transplantation, 15 (63): 1-6 (
1997); Knechtle et al. , Surgary, 124 (2): 438.
-446 (1998); LeMaistre, Clin. Lymphoma, 1: S37
-40 (2000); Martm et al. , J .; Am. Acad. Dermato
l. 45 (6): 871-881, 2001)). DAB389IL-2 (ONTAK
(Registered trademark) is currently the only Food and Drug Administration approved protein fusion toxin, using the cytokine IL-2 that targets cells with DT toxin and L-2 receptor, and skin T cells Approved for the treatment of lymphoma (CTCL) (Figgitt et a
l. , Am. J. et al. Clin. Dermatol. , 1 (1): 67-72 (2000);
Foss, Clin. Lymphoma, 1 (4): 298-302 (2001); Mu
rphy et al. , In Bacterial Toxins: Method a
nd Protocols, Holst O, ed, Humana Press, Tot
owa, N .; J. et al. , Pp. 89-100 (2000)). ONTAK® is variously referred to as Denileukin diftitox, DAB389-IL-2, or Onzar. The structure, in turn, consists of residues methionine, residues 1-386 of natural DT, residues 484-485 of natural DT and residues 2-133 of IL-2 (SEQ ID NO: 148). Therefore,
Full length ONTAK® contains 521 amino acids. As a result of the methionine residue added at the N-terminus of ONTAK®, it is recognized that diphtheria sequence numbering, according to one, is outside the scope of the register having the ONTAK® number. Should be done.

A number of other poisons, particularly ricin toxin and Pseudomonas exotoxin A, have been used to develop both immunotoxins, fusion toxins and chemical conjugates. However, these molecules have not successfully completed clinical trials and all show VLS as a significant side effect (Kre
itman, Adv. Pharmacol. 28: 193-219 (1994); Pu
ri et al. , Cancer Research, 61: 5660-5661 (1
996); Pastan, Biochim Biophys Acta. 24: 133
3 (2): C1-6 (1997); Frankel et al. , Supra (199
7); Kreitman et al. , Current Opin. Invest. D
rugs, 2 (9): 1282-1293 (2001)). Thus, the modifications described herein for diphtheria toxin can be extrapolated to other toxins such as, for example, ricin and Pseudomonas exotoxin A.

In some embodiments, one or more (x) D (y) and / or (x) D (y)
Toxins or compounds modified based on the T motif or its flanking sequences can be used to suppress VLS in vivo. Accordingly, it is contemplated that such mutations affecting (x) D (y) sequences or flanking sequences can alter the ability of a polypeptide to induce or accompany these sequences. In one non-limiting example, diphtheria toxin is modified to suppress VLS in vivo.

To produce a toxin or compound with reduced ability to induce VLS, one or more (up to all and eg all) remaining (x) D (y) and / or (x) D (y )
It is contemplated that the T sequence has a reduced exposure to the surface of the polypeptide. For example, (x) D (y) and / or (x) D () that is at least partially located in an unexposed portion of the polypeptide or shielded from full or partial exposure to the surface of the molecule.
y) It is intended that the T sequence does not interact well with cells, receptors or other molecules that promote or induce VLS. Thus, from the primary structure of the polypeptide, (x) D (y) and / or
Or (x) Complete elimination of D (y) T sequences may not necessarily be necessary to generate a toxin or molecule that has the ability to induce or promote reduced VLS. But all (x
Removal of the D) y (D) and / or (x) D (y) T sequences is intended to produce a composition having the lowest ability to induce or promote VLS.

To see if the mutation is likely to generate a polypeptide with a less exposed (x) D (y) and / or (x) D (y) T motif, it is moved or added. The presumed arrangement of the (x) D (y) and / or (x) D (y) T sequences may be used to transform the mutated sequences into such methods known to those skilled in the art, such as, but not limited to: X-ray crystallography, N
It can be determined by comparison with the secondary and tertiary structure sequences of the unmutated polypeptide, as determined by MR or computer modeling. Computer models of various polypeptide structures are available in the literature and computer databases. In one non-limiting example, the Entrez database website (ncb
i. nlm. nih. gov / Entrez /) can be used to identify target sequences and regions for mutagenesis. The Entrez database, if known,
Interconnected to a database of 3D structures for the identified amino acid sequences. (X) D (y), (x) D (y) in a polypeptide where such a molecular model is exposed more than a similar sequence embedded inside the polypeptide to contact an external molecule. ) Can be used to identify T and / or flanking sequences. (X) D (y), (x) D (y) T and / or flanking sequences that are more exposed to contact external molecules promote VLS and other toxic effects associated with these sequences Or appear to contribute more to inhibition and should therefore be the primary target for mutagenesis. The structure of the mutant or wild type polypeptide may be determined directly by X-ray crystallography or NMR prior to use in in vitro or in vivo assays, as is known to those skilled in the art.

When the amino acid sequence containing the (x) D (y) and / or (x) D (y) T sequence is altered in the polypeptide, the change in its ability to induce or promote at least one toxic effect is Assays can be performed using any of the techniques described herein or known to those of skill in the art.

As used herein, an amino acid sequence containing (x) D (y) or (x) D (y) T sequences is “altered”, “altered”, “altered” and “ A “change” refers to a chemical modification of an amino acid sequence containing (x) D (y) and / or (x) D (y) T sequences within a polypeptide known to those skilled in the art, as well as to such amino acid sequences. Any mutation, including but not limited to insertion, deletion, truncation or substitution. Such a change is (x)
Alter at least one toxic effect (ie, ability to promote VLS, EC damage, etc.) of one or more amino acid sequences containing D (y) and / or (x) D (y) T sequences Or can be modified (suppressed). As used herein, (x) D (
The amino acid sequence containing y) sequence or (x) D (y) T sequence is C-terminal and / or (x) D (y) and / or (x) D (y) T tri- or tetrapeptide sequence Alternatively, it can contain at least one flanking sequence at the N-terminus. Such “changes” can be made in the synthetic polypeptide or in the nucleic acid sequence expressed to produce the mutant polypeptide.

In one embodiment, the change in amino acid sequence containing the (x) D (y) and / or (x) D (y) T sequence is due to removal of the amino acid sequence. As used herein,
“Remove”, “removed”, “remove” or “removal” for an amino acid sequence containing (x) D (y) and / or (x) D (y) T sequence means (x) D (y) and / or (
x) refers to a mutation in the primary amino acid sequence that eliminates the presence of D (y) T tri- or tetrapeptide sequence and / or at least one native flanking sequence. “Removed” or “
The term “lack” is used interchangeably.

One aspect of the present application relates to a genetically modified polypeptide of diphtheria toxin (DT) having reduced binding to human vascular endothelial cells (HUVEC). These modified polypeptides are hereinafter referred to as modified DT, modified DT polypeptide or DT variant. This application is D
Within the (x) D (y) motif of the T polypeptide, ie the native DT sequence (SEQ ID NO: 1
) Residues 6-8 (VDS), residues 28-30 (VDS) and residues 289-291 (IDS)
), Or residues 7-9 (VDS), SEQ ID NO: 2 or 200, residues 29-31 (VD)
S) and modified DT with one or more changes at residues 290-292 (IDS). Since (x) D (y) motifs are referred to as “VLS motifs”, a modified DT polypeptide having one or more modified (x) D (y) motifs is referred to as “VLS modified D
It can be referred to as a “T polypeptide”.

One aspect of the present application relates to genetically modified polypeptides of toxins (eg, diphtheria toxin (DT)) that have reduced binding to human vascular endothelial cells (HUVEC). These modified toxins are hereinafter referred to as modified toxins. This application relates to (x) D / E (y
) Provide modified toxins having one or more changes within the motif. For example, the (x) D / E (y) motif found in DT is the residue 6--6 of the native DT sequence (SEQ ID NO: 1).
8 (VDS), residues 28-30 (VDS) and residues 289-291 (IDS) or SEQ ID NO: 2 or 200 residues 7-9 (VDS), residues 29-31 (VDS )
And residues 290-292 (IDS). (X) D / E (y) motif is "VLS
Because they are referred to as “motifs”, modified toxin polypeptides having one or more modified (x) D / E (y) motifs are sometimes referred to as “VLS modified toxin polypeptides”.

Induces VLS, induces apoptosis and induces other effects (x)
With regard to the identification of D / E (y) and (x) D / E (y) T motifs, the creation of a new family of molecules of VLS inhibitors allows these molecules to exert their greatest beneficial effect. . For example, the reduced toxicity of toxin therapeutics using the compositions and methods disclosed herein allows treating a larger patient population or treating more advanced diseases (eg, cancer). Make it possible to do. In certain embodiments, the modified protein or fusion protein based on (x) D / E (y) and / or (x) D / E (y) T motif or its flanking sequence is VLS or other in vivo Can be used to suppress activity.

(X) Conserved aspartic acid (D) or conserved glutamic acid (E) to produce peptides, polypeptides or proteins lacking D / E (y) and / or (x) D / E (y) T sequences. ) May be deleted or mutated, aspartic acid or glutamic acid may be replaced with another amino acid, or one or more amino acids may be inserted at or adjacent to that position. Modifications contemplated herein include alanine (A), glutamic acid (E), serine (S), isoleucine (as a result of deletion or mutation events).
I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), glycine (G), threonine (T), tryptophan (W), tyrosine (Y), proline ( P), histidine (H), glutamine (Q), asparagine (N),
Substitution of (D) or (E) residues in the sequence by amino acid residues selected from lysine (K), arginine (R) and the modified or unusual amino acids of Table 1.

Alternatively, conserved aspartic acid (D) is deleted to produce a peptide, polypeptide or protein that lacks (x) D / E (y) and / or (x) D / E (y) T sequences. Alternatively, it can be mutated, aspartic acid can be replaced with another amino acid, or one or more amino acids can be inserted at or adjacent to that position. Modifications contemplated herein include isoleucine (I
), Valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine (G), threonine (T), tryptophan (
W), tyrosine (Y), proline (P), histidine (H), glutamine (Q), asparagine (N), lysine (K), arginine (R) and modified or unusual amino acids from Table 1 Substitution of the (D) residue in the sequence with an amino acid residue.

In one embodiment, the (x) residue is (x) D / E (y) and / or (x) D / E.
(Y) can be deleted, substituted or moved by insertion of one or more amino acids to remove T. Modifications contemplated herein include phenylalanine (F), cysteine (C), methionine (M), threonine (as a result of deletion or mutation events.
T), tryptophan (W), tyrosine (Y), proline (P), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), lysine (K), arginine (R), glycine (G), serine (S), alanine (A), leucine (L) and (x) residues in the sequence by amino acid residues selected from the modified or unusual amino acids in Table 1 Includes substitution.

Alternatively, (x) residues are deleted, substituted or moved by insertion of one or more amino acids to remove (x) D (y) and / or (x) D (y) T be able to. Modifications contemplated herein include phenylalanine (F), cysteine (C), methionine (M), threonine (T), tryptophan (W), tyrosine (Y), proline as a result of deletion or mutation events. (P), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), asparagine (N), lysine (K), arginine (R) and the modified or abnormal amino acids in Table 1 Substitution of (x) residues in the sequence by selected amino acid residues. For example, a V or I amino acid residue as described is substituted with any such amino acid residue.

In one embodiment, the (y) residue is (x) D / E (y) and / or (x) D / E
(Y) To remove T, it can be deleted, substituted or moved by insertion of one or more amino acids. Amino acids that can replace a (y) residue in the sequence as a result of a deletion or mutation event are, for example, isoleucine (I); phenylalanine (F); cysteine / cystine (C); methionine (M); alanine ( A); Glycine (G); Threonine (
T); tryptophan (W); tyrosine (Y); proline (P); histidine (H); glutamic acid (E); glutamine (Q); aspartic acid (D); asparagine (N); Arginine (R), leucine (L), valine (V), serine (S) and, for example, but not limited to, the amino acids shown in Table 1.

Alternatively, (y) residues are deleted, substituted or moved by insertion of one or more amino acids to remove (x) D (y) and / or (x) D (y) T sequences. Can be made. An amino acid that can replace a (y) residue in the sequence as a result of a deletion or mutation event is
For example, isoleucine (I); phenylalanine (F); cysteine / cystine (C);
Methionine (M), alanine (A); glycine (G); threonine (T); tryptophan (W), tyrosine (Y), proline (P); histidine (H); glutamic acid (E);
Glutamine (Q); aspartic acid (D); asparagine (N), lysine (K); and arginine (R) and, for example, but not limited to, the amino acids shown in Table 1.

Other residues located in the physical region, three-dimensional space, or near the HUVEC binding site and / or the (x) D / E (y) motif to inhibit, suppress or eliminate VLS Can be mutated or altered. Amino acids targeted for mutation within the flanking region include amino acids at or near the surface of the native toxin protein. Change
(X) D / that can neutralize or reverse the charge in specific areas of the protein surface
Charged residues within the region of the E (y) motif can be removed or replaced. Change is also the physical area,
The size and / or hydrophilicity of the amino acids in the space or near the (x) D / E (y) sequence or in the active site of the protein can also be varied. For example, LDV constitutes the minimal active site of the CS1 domain of fibronectin involved in its binding to the α4β1 integrin receptor (M
akarem and Humphries, 1991; Wayner and Kov
ach, 1992; Nowlin et al. 1993). However, fibronectin (FN) does not damage HUVEC. Instead, FN protects HUVEC from RTA-mediated damage (Baluna et al., 1996). Unlike RTA, FN
It has a C-terminal LDV-flanking proline instead of threonine. In disintegrins, residues adjacent to RGD play a role in ligand binding (Lu et
al. , 1996). The difference between the ability of LDV or homolog-containing molecules to promote vascular integrity (eg, FN) or disrupt it (eg, DT) is due to the orientation of the LDV motif, or interaction (ie, binding) Can depend on the availability of and thus can depend on flanking sequences. Thus, a change in one or more flanking residues of the (x) D / E (y) sequence can increase or decrease the ability of the molecule to promote VLS. And (x) D
Changes exposed on the outer surface of the protein so that the / E (y) sequence interacts with other proteins such as receptors enhances VLS-promoting activity, whereas configurations that are less exposed are VLS-promoting activity Can be reduced.

(X) at least one mutation, chemical modification, migration or other change in the N-terminal or C-terminal flanking sequence of the D / E (y) and / or (x) D / E (y) T sequence is A protein, polypeptide or peptide having the ability to promote reduced VLS can also be produced. Preferably, such mutations or changes will occur at one or more residues that do not affect the active site. In other embodiments, the mutation or change is from about 1, about 2, about 3, about 4, about 5, about 6 or more N-terminal and / or C-terminal (x). It will occur at one or more residues up to the D / E (y) tripeptide sequence. In other embodiments, one or more residues that are not adjacent to the (x) D / E (y) tripeptide may contribute to the function of the (x) D / E (y) motif. Such residues can be identified by their proximity to the tripeptide sequence in a three-dimensional model as described herein and as known to those skilled in the art and considered for variation as part of the flanking sequence. Is done. Such changes are (x) D / E (y) as long as one or more “wild-type” flanking residues are altered, removed, transferred, and chemically modified.
And (x) any of the above changes may be included to alter the D / E (y) T sequence.

Such amino acid modifications can be assayed for the ability to efficiently deliver the catalytic domain of DT to target cells within the context of the fusion protein and not reconstitute the intact VLS motif. Provided herein are modified diphtheria toxins that have a reduced ability to induce VLS. Any remaining (x) D / E (y) and / or (x) D / E (y) T sequences should have reduced exposure to the surface of the protein, polypeptide or peptide, if possible .

In one non-limiting example, (x) D / E () that is at least partially located in an unexposed portion of diphtheria toxin or protected from full or partial exposure to the surface of the molecule.
It is contemplated that y) and / or (x) D / E (y) T sequences will not interact well with cells, receptors or other molecules that promote or induce VLS. Thus, complete elimination of (x) D / E (y) and / or (x) D / E (y) T sequences from the primary structure of diphtheria toxin does not necessarily result in toxins or molecules with reduced VLS inducing or promoting ability. It is intended not to generate. However, in one embodiment, all (x) D / E (y) and / or (
x) The D / E (y) T sequence is removed to produce a composition with the lowest VLS inducing ability.

Mutations are not well exposed (x) D / E (y) and / or (x) D / E (y
(X) D / E (y) and / or (x) D / E (y) T sequences that have been moved or added to see if they are likely to generate modified toxins with T motifs) The presumed configuration of is not mutated of the mutated sequence as determined by such methods known to those skilled in the art, such as, but not limited to, X-ray crystallography, NMR or computer modeling It can be determined by comparison with the secondary and tertiary structure sequences of the (polypeptide) toxin. Computer models for various polypeptides and peptide structures are available in the literature and computer databases. In one non-limiting example, the Entrez database website (www.ncbi.nlm.nih.gov/Entrez/) can be used to identify target sequences and regions for mutagenesis. The Entrez database, if known, is interconnected to a database of 3D structures for identified amino acid sequences. Such molecular models are (x) D / E (y), (x) in diphtheria toxin that are exposed more than a polypeptide or a similar sequence embedded in the interior of a polypeptide to contact an external molecule. x) Can be used to identify D / E (y) T and / or flanking sequences. (X) D / E (y), (x) D / E (y) T and / or flanking sequences that are more exposed to contact external molecules (eg, receptors)
It appears to contribute by promoting or suppressing S and other toxic effects associated with these sequences and should therefore be the primary target for mutagenesis. In certain embodiments, when it is desirable to add at least one (x) D / E (y), (x) D / E (y) T and / or flanking sequences, it contacts an external molecule. The more exposed protein region is therefore preferred as a site for adding such sequences. The structure of the mutant or wild type (polypeptide) toxin could be determined directly by X-ray crystallography or NMR prior to use in an in vitro or in vivo assay, as is known to those skilled in the art. .

The amino acid sequence containing (x) D / E (y) and / or (x) D / E (y) T sequence is (
When altered in a polypeptide) toxin, the change in its ability to promote at least one toxic effect can be assayed using either the techniques described herein or techniques known to those of skill in the art. Methods for changing amino acid sequences are described in more detail below and are known in the art.

Modifications (changes) are substitutions, insertions or deletions of these amino acids that allow modification of unmodified or pre-modified sequences within these regions, but do not impair the cytotoxicity of the toxin or venom. . These modifications are involved in mediating interactions with endothelial cells and thus VDS / ID
It will not contain modifications that regenerate the S sequence. Thus, such native recombination sequences are
It contains a novel series of mutants that maintain the unique function of the unique domain of the toxin while at the same time significantly reducing its ability to interact with vascular endothelial cells.

In one embodiment, the DT variants of the present invention eliminate the motifs associated with VLS, thereby inhibiting the clinical side effects commonly associated with this syndrome (x) D / E (y) Within one of the motifs, ie residues 6-8 (VD of SEQ ID NO: 1
S), residues 28-30 (VDS) and residues 289-291 (IDS), at least 1
Including modifications. However, the modified DT of the present application, when incorporated into a protein fusion toxin, is as effective as DT387 in its ability to facilitate delivery of its catalytic domain into the cytosol of target eukaryotic cells and It is valid. DT387 (SEQ ID NO: 2) is
A truncated DT protein containing amino acid residues 1-386 of the native DT protein containing the catalytic and translocation domains and the addition of an N-terminal methionine. DT
₃₈₉ (SEQ ID NO: 200) is a truncated DT protein containing, in order, residues methionine, residues 1-386 of native DT, and residues 484-485 of native DT. In one embodiment, the DT variants of the present invention eliminate the motifs associated with VLS, thereby inhibiting the clinical side effects commonly associated with this syndrome (x) D / E
(Y) Within one of the motifs, ie residues 7-9 (VDS) of SEQ ID NO: 2 or 200
, Residues 29-31 (VDS) and residues 290-292 (IDS) contain at least one modification.

The unmodified diphtheria toxin can have, for example, the amino acid sequence of SEQ ID NO: 2 or 200 or the amino acid sequence of SEQ ID NO: 4-147. In one embodiment, a modified diphtheria toxin that has cytotoxicity comparable to unmodified diphtheria toxin has substantially similar cytotoxicity as compared to unmodified diphtheria toxin, or at least about 70%, at least about 75. %, At least about 80%, at least about 85%, at least about 90%, or at least about 95% or more of a modified diphtheria toxin. Purified DAB ₃₈₉ IL-2 produced in E. coli is generally 5 × 10 ⁻¹¹ M
This produces an IC ₅₀ between 1 × 10 ⁻¹² M. Thus, in another embodiment, a modified diphtheria toxin having cytotoxicity comparable to unmodified diphtheria toxin is about 5 × 10 ⁻¹¹ M
About 1 × 10 ⁻¹² M, about 1 × 10 ⁻¹⁰ M to about 1 × 10 ⁻¹⁰ M, about 1 × 10 ⁻⁹ M to about 1 × 10 ⁻¹⁰ M, or about 1 × 10 ⁻⁸ M to about Refers to modified diphtheria toxin with an IC ₅₀ between 1 × 10 ⁻⁹ M. The cytotoxicity of modified diphtheria toxin compared to unmodified diphtheria toxin can be tested in a cytotoxicity assay, such as the cytotoxicity assay described in the Examples below.

(X) In addition to modifications in the D / E (y) motif, the modified DT retains the ability to translocate and kill target cells into cells when the cleaved molecule is fused with a cell binding domain. Can further contain deletions or substitutions of 1-30 amino acids, 1-10 amino acids, or 1-3 amino acids of SEQ ID NO: 2 or 200.

In one embodiment, a modified diphtheria toxin, wherein the modified diphtheria toxin has an amino acid sequence as shown in SEQ ID NO: 2 or 200, wherein the amino acid sequence has one or more amino acid modifications therein , Wherein at least one amino acid modification is (x) D in a region such as residues 7-9, 29-31 and 290-292 of SEQ ID NO: 2 or 200, for example.
Provided herein are modified diphtheria toxins that are performed within the motif and have a cytotoxicity that is performed within the motif and the modified diphtheria toxin is comparable to the unmodified diphtheria toxin. In one embodiment, the modification of position (x) is A, S, E, F, C, M, T, W, Y, P, H, Q, D,
N, K, R, G, L, or a modification of Table 1 or substitution of V or I with an unusual amino acid. In one embodiment, the modification at position D is A, S, E, I, V, L, F, C, M, G.
, T, W, Y, P, H, Q, N, K, R, or a modification of Table 1 or substitution of D with an abnormal amino acid. In one embodiment, the modification at position (y) is I, F, C, M, A, G,
T, W, Y, P, H, E, Q, D, N, K, R, S, L, V and the modifications in Table 1 or substitution with abnormal amino acids.

Alternatively, in another embodiment, a modified diphtheria toxin having one or more amino acid modifications therein, wherein at least one amino acid modification is SEQ ID NO: 2 or 200
(X) D of a region selected from the group consisting of residues 7-9, 29-31 and 290-292 of
A modified diphtheria toxin having one or more amino acid modifications therein, wherein the modified diphtheria toxin is performed within the / E (y) motif and has a cytotoxicity comparable to the unmodified diphtheria toxin, Provided. In one embodiment, the modification at position (x) is F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, or the modification or unusual amino acid of Table 1 Substitution of V or I by In another embodiment, the modification of position D / E is I,
V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R, or a modification of Table 1 or substitution of D / E with an abnormal amino acid. In one embodiment, the modification at position (y) is I, F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K, R, S, L, V
And Table 1 modification or substitution with unusual amino acids.

In one embodiment, the modified diphtheria toxin is V7T, V7N, V7D, D8N,
S9A, S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31
N, 1290T, D291E, S292A, S292G and one or more modifications selected from S292T.

In one embodiment, the modified diphtheria toxin contains two modifications. Such modified diphtheria toxins are combinations of mutations such as V7N V29N, V7N V29.
T, V7N V29D, V7T V29N, V7T V29T or V7T V29D may be included.

In one embodiment, the modified diphtheria toxin contains three modifications. Such modified diphtheria toxins are combinations of mutations such as V7N V29N I290N, V7N.
V29N I290T, V7N V29N S292A, V7N V29N S292
T, V7N V29T I290N, V7N V29T I290T, V7N V29T
S292A, V7N V29T S292T, V7T V29T I290T, and the like.

About one, two, three, four, five, six, seven, eight, nine, ten, 11 in one or more (x) D / E (y) motifs Modified diphtheria toxins containing 12, 12, 13, 15, 15, 16, 17, 18, 19, or up to about 20 modifications use the methods described herein Thus, amino acid residues are prepared by serial modification and comparison of activity after each modification to unmodified or pre-modified diphtheria toxin. Alternatively, a modified diphtheria toxin comprising two or more modifications uses the methods described herein to simultaneously modify two or more amino acid residues and compare the activity to a previously unmodified diphtheria toxin. It is prepared by. Modified toxins can be tested for activity using assays known in the art and described herein, such as, but not limited to, cytotoxicity assays and ADP ribosylation assays.

Expressed toxin-mutants and toxin-fusion proteins can be tested for their functional activity. Methods for testing the activity of various toxins are well known in the art. For example, D
The VLS effect of T variants and DT-fusion proteins is shown in HU as described in Example 2
Can be tested in VEC. The ribosyltransferase activity of a DT variant or DT-fusion protein can be tested in the ribosyltransferase assay described in Example 3. The cytotoxicity of the DT variant or DT-fusion protein can be tested as described in Example 4. VLS-modified DT fusion toxins using these ligands are useful for treating cell types of cancer or other diseases where specific binding exists. Toxins with modifications of T cell epitopes, B cell epitopes and VLS motifs Methods for reducing immunogenicity by modification of T cell and B cell epitopes and reduction of vascular leakage syndrome by modification of VLS motifs as described herein Besides how to make
Toxins and modified toxins have reduced immunogenicity (T cells, B cells, or both) and reduced ability to cause vascular leakage syndrome (ie, reduced binding to endothelial cells and vascular endothelial cells as well as endothelial cells) Both modifications can be included to produce polypeptides that exhibit reduced interstitial disruption and other indications of vascular leak syndrome as described herein.

In order to generate toxins with modifications in T cell epitopes, B cell epitopes, VLS motifs and combinations thereof (eg, diphtheria toxins), modifications of the amino acid residues made for each type of modification Considered in the light of qualification. In one non-limiting example, modification of both T cell epitopes and VLS motifs is desirable. When modifying both T cell epitopes and VLS motifs, modification of at least one amino acid within at least one T cell epitope within the toxin should not create a VLS motif. Similarly, modification of at least one amino acid within at least one VLS motif within the toxin should not create a T cell epitope. T cell epitopes or V
Modification of the LS motif should not reinduce pre-modified T cells or VLS motifs. Furthermore, modifications of such polypeptides can also take into account modifications of B cell epitopes that should not create or reinduce T cell epitopes or VLS motifs, if desired.

Modifications that affect the cytotoxicity of the toxin can also be made. For example, when one or more modifications are made to one or more T cell epitopes, a VLS motif and / or B cell epitope is prepared and the cytotoxicity of the modified toxin is lower than that of the unmodified toxin In
It is contemplated herein that one or more modifications can be made to restore cytotoxicity to a level comparable to that of unmodified toxin or to an effective level.

The membrane insertion domain (translocation domain) of diphtheria toxin (T) contains an amphipathic region responsible for delivery of the catalytic domain (C) domain to the cytosol of the cell. In one non-limiting example, substitution of one or more amino acid residues in the amphiphilic region with one or more different amino acid residues that retain the helical structure of the region can retain cytotoxicity. A discussion of the composition and distribution of charged and hydrophobic amino acid residues and their modifications within the amphiphilic region is also contemplated herein. Examples of charged amino acid residues include Glu,
Asp, Asn, Gln, Lys, Arg and His are mentioned. Hydrophobic amino acids include, but are not limited to, alanine and phenylalanine.

Modification of the binding gap of the catalytic domain of the ADP-ribosylated protein family described herein can also affect cytotoxicity. In one non-limiting example, modification of one or more amino acid residues of the F / YXSTX motif of the diphtheria toxin C domain can affect the cytotoxicity of the polypeptide. . Substitution of one or more amino acid residues in this region with one or more different amino acid residues that retain the catalytic domain can retain cytotoxicity. ADP-ribosylated protein family [
For example, comparison of Pseudomonas aeruginosa exotoxin A] with related constituent members can provide guidance on amino acid residue modifications and composition of domains such as the diphtheria toxin C domain. Modifications such as these are also contemplated herein.

Provided herein are methods for preparing modified toxins having reduced immunogenicity, reduced VLS effects (ie, reduced endothelial cell binding) and combinations thereof. Also contemplated within the scope of the invention described herein are methods for selecting modified toxins having reduced immunogenicity, reduced VLS effects, and combinations thereof.

In one embodiment, the toxin is modified for T cell epitopes, B cell epitopes, VLS motifs and combinations thereof, and the amino acid sequence of the modified toxin is for the generation or reintroduction of T cell epitopes, B cell epitopes or VLS motifs. Continuously examined for. When it is observed that a T cell epitope, a B cell epitope, a VLS motif, or a combination thereof has been generated or reintroduced, the amino acid residues therein are the above T cell epitope, B cell epitope, VLS motif. Or, in order to remove these combinations, it is further modified without generating or reintroducing any T cell epitope, B cell epitope, VLS motif or combination thereof.

In another embodiment, T cell epitopes within the toxin are first identified and modified, and then any VLS motif within the toxin is identified and modified. The modified toxin is then examined for the generation and reintroduction of any T cell epitope, and any such T cell epitope is modified without generating or reintroducing the VLS motif. In yet another embodiment, the VLS motif in the toxin is first identified and modified, and then the T cell epitope in the toxin is identified and modified.

Application of the methods described herein also provides methods for preparing modified toxins that exhibit reduced immunogenicity, reduced VLS effects (ie, reduced endothelial cell binding), and combinations thereof. Also contemplated within the scope of the invention described herein are methods for selecting modified toxins having reduced immunogenicity, reduced VLS effects, and combinations thereof. Methods for testing modified toxins for such characteristics are known in the art and are described, for example, in the examples provided herein. In one embodiment, a method of preparing a modified toxin that exhibits reduced immunogenicity and reduced VLS effect compared to an unmodified toxin identifies at least one T cell epitope within the amino acid sequence of the toxin, and at least Modify at least one amino acid residue within one identified T cell epitope, identify at least one VLS motif within the amino acid sequence of the toxin, and at least within at least one identified VLS motif It consists of modifying one amino acid.

In another embodiment, a modified toxin that exhibits reduced immunogenicity and reduced VLS effect relative to an unmodified toxin identifies at least one T cell epitope within the amino acid sequence of the toxin and at least one identification Modifying at least one amino acid residue within the identified T cell epitope, identifying at least one VLS motif within the amino acid sequence of the toxin, and at least one amino acid within the at least one identified VLS motif Produced by a method of modifying.

In yet another embodiment, reduced immunogenicity and reduced VL compared to unmodified toxin
The method of selecting a modified toxin exhibiting an S effect identifies at least one T cell epitope within the amino acid sequence of the toxin and at least one within at least one identified T cell epitope.
Modified at least one amino acid residue, identified at least one VLS motif within the amino acid sequence of the toxin, modified at least one amino acid within the at least one identified VLS motif and compared to the unmodified toxin Selecting a modified toxin that exhibits reduced immunogenicity.

VI. Toxin As used herein, the term “toxin” refers to any anti-cell agent, including but not limited to any combination of cytotoxin and / or anti-cell agent. In some embodiments,
Toxins include, for example, plant toxins, fungal toxins, bacterial toxins, ribosome inactivating proteins (RI
P) or a combination thereof. Toxins include, but are not limited to, abrin A
Chain, diphtheria toxin (DT) A chain, Pseudomonas exotoxin, RTA, Shiga toxin A chain, Shiga-like toxin, gelonin, momordin, porkweed antiviral protein, saporin, trichosanthin, barley toxin and various known in the art Other toxins. The toxins used herein are specifically staphylococcal enterotoxin toxin B (SE
B) Exclude hirudin and bouganin proteins.

Diphtheria toxin is a member of the mono-ADP-ribosylating toxin family, and the mono-ADP-ribosylating toxin family further includes cholera toxin, Pseudomonas exotoxin A
And toxins such as pertussis toxin and clostridial C3-like toxin. Members of this family contain a number of similar protein domains and motifs, particularly the catalytic site of the toxin. For example, the catalytic sites of many members of this family are known to contain glutamate residues that are important in the catalytic function of these toxins. Members of this family are considered to be within the scope of the invention described herein using DT as a typical toxin. DT consists of three domains: a catalytic domain, a transmembrane domain, and a receptor binding domain (Choe et al., Nature, 357: 216-222 (199).
2)). The nucleic acid and amino acid sequences of native DT are shown in Greenfield et a
l. , PNAS (1983) 80: 6853-6857. Native DT targets cells expressing heparin-binding epidermal growth factor-like receptor (Naglis
h et al. , Cell, 69: 1051-1061 (1992)). First generation targeted toxins were initially developed by chemically cross-linking novel target ligands to toxins such as DT or mutants of DT that lack cellular binding (eg CRM45) ( Cawley, Cell, 22: 563-570 (1980); Bacha et a
l. , Proc. Soc. Exp. Biol. Med. , 181 (1): 131-138.
(1986); Bacha et al. , Endocrinology, 113 (3)
: 1072-1076 (1983); Bacha et al. , J .; Biol. Che
m. 258 (3): 1565-1570 (1983)). Natural cell binding domains or bridging ligands that direct DT poisons to receptors on the surface of certain classes of receptor bearing cells are:
Must have an intact catalytic domain and a translocation domain (C
awley et al. , Cell, 22: 563-570 (1980);
rSpek et al. , J .; Biol. Chem. , 5: 268 (16): 1207
7-12082 (1993); vanderSpec et al. , J .; Biol. C
hem. , 7 (8): 985-989 (1994); vanderSpec et al.
. , J .; Biol. Chem. , 7 (8): 985-989 (1994); Roscon
i, J. et al. Biol. Chem. , 10; 277 (19): 16517-161278 (2
002)). These domains are important for target cell delivery and addiction after receptor internalization (Greenfield et al., Science, 238 (4826)).
536-539 (1987)). Once the toxin, toxin complex or fusion toxin binds to the cell surface receptor, the cell harbors the toxin-coupled receptor through endosite vesicles. As the vesicles are processed, they are acidified and the translocation domain of the DT venom carries a structural rearrangement that inserts nine transmembrane segments of the toxin into the membrane of the endosite vesicle. This event causes the formation of productive pores through which the catalytic domain of the toxin passes. Once translocated, the catalytic domain with ADP-ribosyltransferase activity is released into the cytosol of the target cell where it is free for toxic translation and hence cell death (v
andSpeck et al. , Methods in Molecular Bi
theory, Bacterial Toxins: methods and Proto
cols, 145: 89-99, Humana press, Totowa, N .; J. et al. ,
(2000)).

Less than 10 molecules of DT will kill cells if they enter the cytosol (but the invasion process is inefficient, so many times that number must bind to the cell surface). Must). This surprising ability initially raises concerns that such poisons are too strong and uncontrollable. However, toxins such as DT can be easily detoxified by removing or modifying their cell binding domains or subunits (except when directed to target cells). The remaining portion of the toxin (which lacks the cell binding domain) is then linked to a ligand (eg, a polypeptide containing the cell binding domain or portion thereof) that targets the toxin portion to the target cell. By selecting a polypeptide or portion thereof that contains a cell binding domain that lacks undesired cross-reactivity, the fusion protein is safer and less non-specific than most conventional anticancer drugs Has a cytotoxic effect. Another major attraction of toxins such as DT is that they kill killing cells as effectively as the cells in which they are dividing because these toxins are inhibitors of protein synthesis. Thus, tumors or infected cells that are not in cycle at the time of treatment are subject to fusion protein cytotoxicity.

Toxins such as DT often contain two disulfide-linked chains, an A chain and a B chain. The B chain has both a cell binding region and a translocation region, facilitating the insertion of the A chain into the cytosol through the membrane of the acidic cell compartment. The A chain in this case kills the cell after translocation. For its in vivo use, the ligand and toxin are linked in such a way as to remain stable while passing through the bloodstream and tissues, but not in the target cell so that the toxin moiety can be released into the cytosol. It is stable.

However, it may be desirable from a pharmacological point of view to use the smallest molecule that can nevertheless provide a suitable biological response. Thus, it may be desirable to use smaller A chain peptides or other toxins that will provide an appropriate anti-cellular response.

The diphtheria toxin described herein contains the amino acid sequence shown in SEQ ID NO: 2 or 200. Diphtheria variants are also known to contain insertions, deletions and / or substitutions of nucleic acid residues in their nucleic acid sequences while still retaining their biological activity. Variants of diphtheria toxin have been characterized to demonstrate nucleic acid mutations among diphtheria toxins (Holmes, RK, J. Infect. Dis., 181 (Supp. 1): S
156-S167 (2000)), and therefore diphtheria toxin can contain various nucleic acid and / or amino acid sequences. Nucleic acid residue insertions, deletions and / or substitutions can also affect the amino acid sequence. However, not all nucleic acid residue changes will result in changes at the amino acid residue level of the protein due to the duplication of the genetic code. Diphtheria toxin nucleic acid and / or amino acid changes (ie, insertions, deletions and / or
Or substitution) is also included within the definition of diphtheria toxin and is contemplated herein. As used herein, diphtheria toxin contains the amino acid sequence shown in SEQ ID NO: 2 or 200 and is further about 90%, 91%, 9 relative to SEQ ID NO: 2 or 200.
Includes diphtheria toxins containing amino acid sequences of 2%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. DT C-terminal truncation can also be performed, for example about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about DT389 or DT387, About 9, about 10, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 Deletion of one amino acid residue. For example, as used herein, diphtheria toxin is SEQ ID NO:
About 90%, 91%, 92%, 93%, comprising the amino acid sequence set forth in amino acid residue 1-382 of 2 or 149 and further to amino acid residue 1-382 of SEQ ID NO: 2 or 149,
Includes diphtheria toxins containing 94%, 95%, 96%, 97%, 98% or 99% homologous amino acid sequences. It will be appreciated that variants of diphtheria toxin can be modified and tested for function using any of the methods described herein.

In one embodiment, a toxin as used herein contemplates a fusion protein between a toxin (eg, diphtheria toxin) and a non-toxin polypeptide that contains at least one cell binding domain. In one non-limiting example, diphtheria toxin or fragment thereof is fused to the cell binding domain of interleukin 2 (IL-2), thus creating a fusion toxin. As described in further detail herein, a fusion protein toxin can also contain linker polypeptides and complexes. Such toxins are also contemplated as toxins that are modified by the inventive methods disclosed herein.

In one embodiment, the toxin is a fusion protein containing a modified toxin, wherein the toxin binding domain is replaced with the binding domain of a non-toxin polypeptide. In another embodiment, the toxin is a fusion protein consisting of a modified diphtheria toxin and a non-toxin polypeptide. In another embodiment, the toxin is a fusion protein consisting of diphtheria toxin and IL-2.

Although diphtheria toxin is often discussed as a representative toxin, it will be appreciated that the methods of the invention described herein are applicable to any of the aforementioned toxins.

VII. Fusion proteins The present invention also provides modified toxin fusion proteins. The modified toxin polypeptide can be fused to a non-toxin polypeptide, for example. In one embodiment, the non-toxin polypeptide is a cell specific binding ligand. The specific binding ligand used in the present invention can contain the entire ligand, or contain the portion of the ligand that contains the entire binding domain of the ligand, or contain the effective portion of the binding domain. Most desirably it contains all or most of the binding domain of the ligand molecule. In one non-limiting example, a DT fusion protein contains a DT-related polypeptide (eg, a modified DT described herein) and a non-DT polypeptide fused in-frame to each other. A DT-related polypeptide corresponds to all or part of a DT variant with reduced immunogenicity or modified T cell epitopes, reduced binding to human vascular endothelial cells, or a combination thereof. In one embodiment, the DT fusion protein contains at least a portion of a modified DT sequence set forth in one of SEQ ID NOs: 4-147. In another embodiment, DT
The fusion protein contains at least one T cell epitope modification. In yet another embodiment, the DT fusion protein contains at least one modification of a T cell epitope and at least one modification set forth in one or more of SEQ ID NOs: 4-147. In yet another embodiment, the DT fusion protein comprises at least one T cell epitope modification;
At least one modification set forth in one or more of SEQ ID NOs: 4-147, and at least one
Contains B cell epitope modifications.

In one embodiment of the compounds disclosed herein, the modified toxin is a fusion toxin in which the non-diphtheria toxin polypeptide is a cell binding ligand. In another embodiment, the cell binding ligand is an antibody or antigen binding fragment thereof, cytokine, polypeptide, hormone,
Growth factor or insulin. In yet another embodiment, the cytokine is IL-2.
It is.

In another embodiment, the modified toxin is a fusion toxin wherein the cell binding domain is an antibody or antigen binding fragment thereof. The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a humanized antibody, a recombinant antibody or a transplanted antibody. The antigen-binding fragment is, for example, Fa
_{b, Fab 2, F (ab} ') 2, scFv, scFv2 ( head of two scFv molecules of the A-chain - tandem coupling tail) can be a single chain binding polypeptides, VH or VL. Methods for preparing antigen-binding fragments are known in the art and are incorporated herein. Useful antibodies include antibodies that can specifically bind to receptors or other moieties expressed on the surface of target cell membranes or tumor-bound antigens.

“Specifically binds” means that the binding agent binds to an antigen on the surface of the target cell with greater affinity than it binds an unrelated antigen. Such affinity is preferably at least about 10 times greater, at least about 100 times greater, or at least about 1000 times greater than the affinity of the binding agent for unrelated antigens. The terms “immunoreactive” and “specifically bind” are used interchangeably herein. In certain embodiments, the anti-tumor antibody or antigen-binding fragment thereof (eg, scFv, scFv2, etc.) recognizes tumor cell surface determinants and is taken up into these cells by receptor-mediated endocytosis. It is. In another embodiment, the antibody or antigen-binding fragment thereof binds to a B cell surface molecule, eg, the B cell surface molecule is CD19 or CD22. Alternatively, the antibody or antigen-binding fragment thereof binds to the ovarian receptor MISIIR (Mueller inhibitor type II receptor).

Cell specific binding ligands include, but are not limited to, polypeptide hormones, for example, polypeptide hormones created using the binding domain of α-MSH selectively bind to melanocytes. And enables the assembly of improved t-MSH chimeric toxins useful for the treatment of melanoma (Murphy, JR et a
l. , Proc. Natl. Acad. Sci. USA. , 83 (21): 8258-8
262 (1986)). Other specific binding ligands that can be used include insulin,
Examples include somatostatin, interleukins I and III, and granulocyte colony stimulating factor. Other useful polypeptide ligands with cell-specific binding domains are follicle stimulating hormone (specific for ovarian cells), luteinizing hormone (specific for ovarian cells), thyroid stimulating hormone (specific for thyroid cells), vasopressin (Specific for uterine cells, and bladder and intestinal cells), prolactin (specific for breast cells) and growth hormone (specific for certain bone tissue cells). Specific binding ligands that can be used include cytokines such as, but not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL
-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-
14, IL-15, β-interferon, α-interferon (INF-α), IN
F-γ, angiostatin, thrombospondin, endostatin, METH-1, ME
TH-2, GM-CSF, G-CSF, M-CSF, tumor necrosis factor (TNF), SVEG
F, TGF-β, Flt3 and B cell growth factor. IL-2 is of particular importance because of its role in allergic reactions with activated T cells and autoimmune diseases such as systemic lupus erythematosus (SLE). Toxin fusion protein produced using B cell growth factor is an immunosuppressant that kills proliferating B cells, has a B cell growth factor receptor and is involved in hypersensitivity reactions and organ rejection Can be used as an agent. Other cytokines include substance P (Benoliel et al., Pain, 79 (
2-3): 243-53 (1999)), VEGF (Hotz et al., J. Ga)
straintest Surg. 6 (2): 159-66 (2002)), IL-3
(Jo et al., Protein Exp Purif., 33 (1): 123-
33 (2004)) and GM-CSF (Frankel et al., Clin. Ca).
ncer Res. , 8 (5): 1004-13 (2002)). Modified fusion toxins using these ligands are useful for treating cancer or other diseases of cell types in which specific binding exists.

In IL-2, the LDL sequence (“VLS” motif) of residues 19-21 (SEQ ID NO: 3) is located in the α-helix and is partially exposed. Asp- of LDL motif
Mutating 20 to Lys eliminates IL-2 binding to the β chain of the IL-2 receptor and subsequent T cell proliferation (Collins et al., 1988). IL-2
Have been reported to directly increase the permeability of vascular endothelium to albumin in vitro and this effect can be suppressed by anti-IL-2 receptor monoclonal antibodies (Dow
nie et al. 1992). LDL sequences in IL-2 damage HUVEC. Asp-20 in LDL of IL-2 is involved in receptor binding and functional activity (Col
ins et al. 1988). Thus, in certain embodiments, mutations in the (x) D / E (y) and / or flanking sequences of IL-2 should be conservative with respect to Asp-20 in order to eliminate or suppress VLS. It is contemplated that it may or may reduce the biological activity of IL-2.

For many cell-specific ligands, the region within each such ligand in which the binding domain is located is now known. Also, advances in solid phase polypeptide synthesis should allow those skilled in the art to synthesize various fragments of the ligand and label these fragments using conventional methods known in the art such as an ELISA assay. By testing for the ability to bind to a class of cells, it is actually possible to determine the binding domain of any such ligand. Thus, the chimeric gene fusion toxin of the present invention need not necessarily contain a complete ligand, but rather can contain only a fragment of a ligand that exhibits the desired cell binding ability. Similarly, cell binding regions with ligand analogs or small sequence changes thereof can be synthesized, tested for their ability to bind to cells, and incorporated into the mixed components of the present invention. The amino acid sequence of a cell binding polypeptide can be analyzed for one or more VLS motifs, T cell epitopes, B cell epitopes, or combinations thereof, and can be modified according to the concepts described herein.

In one embodiment, the toxin fusion proteins of the invention are produced by standard recombinant DNA techniques. For example, DNA fragments encoding various polypeptide sequences can be obtained according to conventional methods, for example, blunted or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, as necessary. They are ligated together in frame by using sticky end filling, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques, such as an automated DNA synthesizer. Alternatively, PCR amplification of a gene fragment can be performed using an anchor primer that is subsequently annealed and re-amplified between two consecutive gene fragments that can yield a chimeric gene sequence. Produces a complementary overhang.

When required for proper conformational folding of the fusion protein, the peptide linker sequence is at a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structure, It can be used to separate toxin-related polypeptides from non-toxin polypeptide components. Such peptide linker sequences can be incorporated into fusion proteins using standard techniques well known in the art and include the following factors: (1) the ability to adopt its flexible extended conformation; (2) The inability to adopt secondary structures capable of interacting with functional epitopes on the surface of toxin-related and non-toxin polypeptides, and (3)
Selection can be based on hydrophobicity or lack of charged residues that can react with the polypeptide functional epitope. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids such as Thr and Ala can also be used in the linker sequence. The amino acid sequence that can be effectively used as a linker is not limited to the following, but Maratea et al. Gene, 40: 39-46,19.
85; Murphy et al. , Proc. Natl. Acad. Sci. USA,
83: 8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,7.
Examples include the amino acid sequences disclosed in Japanese Patent No. 51,180. The linker sequence can generally be about 1 to about 50 amino acids in length. A linker sequence may not be required if the toxin-related and non-toxin polypeptides have a nonessential N-terminal amino acid region that can be used to separate functional domains and suppress steric hindrance.

Chemical crosslinking or conjugation results in various molecular species corresponding to the reaction products, and typically only a small fraction of these products is catalytically and biologically active. In order to be biologically active, the reaction products must be conjugated in a manner that does not inhibit the intrinsic structure and activity of the catalytic and translocation domains of the venom. Separation of active or highly active species from inactive species is not always possible because reaction products often have similar biophysical characteristics including, for example, size, charge density and relative hydrophobicity. Not always. Isolation of large quantities of pure clinical grade active products from chemically cross-linked toxins is typically economical for the production of pharmaceutical grade products for clinical trials and subsequent introduction into the clinical market. It is worth noting that it is not possible. To circumvent this problem, a gene DT protein fusion toxin is created in which the native DT receptor binding domain is genetically replaced with melanocyte stimulating hormone as a surrogate receptor targeting domain (Murphy et al., P
NAS, 83: 8258-8262 (1986)). This approach was DAB48 that was specifically cytotoxic only to those cells that expressed the high affinity form of the IL-2 receptor.
Used with human IL-2 as a surrogate targeting ligand to create 6IL-2 (
Williams et al. , Protein Eng. , 1: 493-498 (1
987)). Subsequent work on DAB486IL-2 showed that cleavage of the 97 amino acid from the DT portion of the molecule resulted in a more stable and more cytotoxic version of DAB389IL-2 than the IL-2 receptor targeted toxin. (Williams et al
. , J .; Biol. Chem. , 265: 11885-889 (1990)). The original structure (486) still had a portion of the native DT cell binding domain. DAB389
The version contains the DT C and T domains with the DT portion of the fusion protein ending in a random coil between the T domain and the associated receptor binding domain. Since then, many other targeting ligands have been genetically fused to this DT poison, DAB389 (VanderSpec et al., Method in Molecule).
ar Biology, Bacterial Toxins: Methods and
Protocols, 145: 89-99, Humana Press, Totowa,
N. J. et al. (2000)). Similar approaches are currently used with other bacterial proteins, and gene fusion toxins are often easier to produce and purify.

VIII. Nucleic acid, vector and host cell Another aspect of the present invention relates to a vector containing a polynucleotide (nucleic acid, DNA) encoding a modified DT variant or a fusion protein thereof.

Nucleotide and polypeptide sequences for various genes have been previously disclosed and can be found in computer databases known to those skilled in the art. One such database is the National Center for Biotechnology Information.
Center of Biotechnology Information) Ge
nbank database and GenPept database. The coding regions of these known genes can be amplified and / or expressed using the techniques disclosed herein or by any technique known to those skilled in the art. Polypeptide sequences can also be obtained by methods known to those skilled in the art, such as automated polypeptide synthesizers such as Applied Biosy.
It can be synthesized by polypeptide synthesis using an automated polypeptide synthesizer available from stems (Foster City, Calif.).

As used herein, “expression vector or construct” means any type of genetic construct that contains a nucleic acid that encodes a gene product capable of transcribing part or all of a nucleic acid coding sequence. A transcript can be translated into a protein but need not necessarily be translated. Thus, in certain embodiments, expression includes both transcription of a gene and translation of RNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid, eg, to create an antisense construct.

Particularly useful vectors are considered to be vectors that are placed under the transcriptional control of a promoter, regardless of whether the coding portion of the DNA segment encodes a full-length protein, polypeptide or smaller peptide. A “promoter” is a DN recognized by a cell synthesizer or an introduced synthesizer that is required to initiate specific transcription of a gene.
Refers to the A sequence. The terms “operably placed”, “under control” or “under transcriptional regulation”
It means that the promoter is in the correct arrangement and orientation with respect to the nucleic acid encoding the gene product to control RNA polymerase initiation and gene expression.

A promoter refers to a 5 ′ non-coding sequence located upstream of a coding segment or exon, eg, recombinant cloning and / or PC, in connection with the compositions disclosed herein.
Since it can be obtained by isolation using the R method, it can be in the form of a promoter necessarily associated with the gene.

In another embodiment, it is contemplated that certain advantages may be obtained by placing the coding DNA segment under the control of a recombinant or heterologous promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a gene in its natural environment. Such promoters include promoters typically associated with other genes, and / or promoters isolated from any other bacteria, virus, eukaryotic cell, or mammalian cell, and / or “naturally Mention may be made of promoters prepared by hand that are not "present", i.e. promoters containing different elements from different promoters or containing mutations that increase, decrease or alter expression.

A promoter is selected for expression that efficiently directs the expression of the DNA segment in the cell type, tissue, or even animal. The use of promoter and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology. For example, S
ambrook et al. , (1989) (incorporated herein by reference). The promoter used can be constitutive or inducible and can be used under conditions suitable to direct high level expression of the introduced DNA segment, eg for large scale production of recombinant proteins or peptides. Can be used under good conditions.

At least one module in the promoter generally serves to position the start site for RNA synthesis. The best known example of this is the TATA box,
In some promoters lacking the TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late gene, a separate element covering the start site itself can fix the location of the start. Useful.

Additional promoter elements regulate the frequency of transcription initiation. Typically, these promoter elements are located in a region 30-110 base pairs (bp) upstream of the start site, although many promoters may further contain functional elements downstream of the start site. It has been revealed. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50 bp away before activity begins to decline. Depending on the promoter, the individual elements appear to function in concert or independently to activate transcription.

The particular promoter used to regulate nucleic acid expression is not considered critical as long as the nucleic acid can be expressed in the target cell. Thus, when human cells are targeted, it is preferable to place a nucleic acid coding region adjacent to a promoter that can be expressed in the human cell and place the nucleic acid coding region under its control. Generally speaking, such promoters may contain human promoters or viral promoters.

In expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not considered critical to the successful implementation of the present invention, and any such sequence can be used. Polyadenylation signals include, but are not limited to, SV40 polyadenylation signals and bovine growth hormone polyadenylation signals that are convenient and known to function well in various target cells. Also intended as an element of the expression cassette is a terminator sequence. These elements can help to increase message levels and minimize reading from the cassette to other sequences.

Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG start codon and adjacent sequences. Exogenous translational control signals,
For example, an ATG start codon may need to be provided. One skilled in the art could easily determine this and provide the necessary signal. It is well known that the start codon must be “in frame” with an open reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be natural or synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer elements.

Cells in which the polypeptide can be expressed simultaneously with other selected proteins, in which case the protein can be expressed simultaneously in the same cell or the gene already has another selected protein It is intended to be able to provide Co-expression consists of two different recombinant vectors, each with either copy of the respective DNA
Can be achieved by transferring the cells simultaneously. Alternatively, a single recombinant vector is
Both proteins can be assembled to contain coding regions, which can then be expressed in cells transfected with a single vector. In any event, the term “co-expression” herein refers to the expression of both a gene and other selected proteins in the same recombinant cell.

As used herein, the terms “engineered” and “recombinant” cells or host cells are intended to refer to cells into which an exogenous DNA segment or gene, eg, a gene encoding a cDNA or protein, has been introduced. To do. Thus, engineered cells can be distinguished from naturally occurring cells that do not contain recombinantly introduced exogenous DNA segments or genes. Thus, an engineered cell is a cell that has a gene or gene introduced by the hand of man. Recombinant cells include cells that have introduced cDNA or genomic genes, and also include genes placed adjacent to promoters that are not necessarily associated with a particular introduced gene.

To express a recombinant polypeptide according to the present invention, whether modified or wild type, one or more expression vectors containing a nucleic acid encoding a wild type or modified protein Will be prepared under the control of the promoter. In order to place the coding sequence “under the control of” the promoter, the 5 ′ end of the transcription start site of the transcription reading frame is generally between about 1 and about 50 “downstream” (ie, 3 ′) of the selected promoter. Place between nucleotides. The “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded recombinant protein. This is the meaning of “recombinant expression” in this context.

Many standard techniques are available for assembling expression vectors containing appropriate nucleic acids and transcriptional / translational control sequences to achieve expression of proteins, polypeptides or peptides in a variety of host expression systems. The types of cells that can be used for expression include, but are not limited to, bacteria such as E. coli and Bacillus subtilis transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors.

Some examples of prokaryotic hosts are E. coli strain RR1, E. coli LE392, E. coli B, E. coli X1776 (ATCC No. 31537) and E. coli W3110 (F-, λ-, prototrophic, ATCC No. 273325), Bacillus subtilis And other enteric bacteria such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. Vectors usually have a replication site and a labeling sequence that can provide phenotypic selection in transformed cells. For example, E. coli is often transformed using a derivative of pBR322 (a plasmid derived from an E. coli species). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides an easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage, must also contain or be modified to contain a promoter that the microorganism can use for expression of its own protein.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors in connection with these hosts. For example, phage λGE
M ™ -11 can be utilized to prepare recombinant phage vectors that can be used to transform host cells such as, for example, E. coli LE392.

Another useful vector is glutathione S for later purification and separation or resolution.
-PIN vectors (Inouye et al., 1985) and pGEX vectors for use in generating transferase (GST) soluble fusion proteins. Other suitable fusion proteins are those having β-galactosidase, ubiquitin, and the like.

The most commonly used promoters for recombinant DNA assembly include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. Although these are most commonly used, other microbial promoters have been discovered and utilized, details regarding their nucleotide sequences have been published, and those skilled in the art will operably link them with plasmid vectors. Make it possible.

The following details regarding recombinant protein production in bacterial cells such as E. coli are provided as typical information on general recombinant protein production, and its adaptation to specific recombinant expression systems is known to those skilled in the art. I will.

Bacterial cells containing expression vectors, such as E. coli, can be in any of a number of suitable media,
For example, grow in LB. Recombinant protein expression can be induced, for example, by adding IPTG to the medium or by switching the incubation to a higher temperature. After culturing the bacteria for an additional period of time, typically 2-24 hours, the cells are harvested by centrifugation and washed to remove residual media.

The bacterial cells are then lysed, for example by disrupting with a cell homogenizer,
Centrifuge to separate concentrated inclusion bodies and cell membranes from soluble cell components. This centrifugation can be carried out under conditions where the concentrated inclusion bodies are selectively concentrated by incorporation of sugars, such as sucrose, into the buffer and centrifugation at a selected speed.

If the recombinant protein is expressed in inclusion bodies, as is often the case, these are washed with any of several solutions to remove some of the contaminating host protein, and then the reducing agent,
For example, in the presence of β-mercaptoethanol or DTT (dithiothreitol), it is solubilized in a solution containing a high concentration of urea (eg 8M) or a chaotropic agent such as guanidine hydrochloride.

It is contemplated that a polypeptide produced by the methods described herein can be overexpressed, i.e., expressed at an increased level relative to its natural expression in a cell. Such overexpression can be assessed by a variety of methods including radiolabeling and / or protein purification. However, a simple and direct method is preferred, for example S
Preferred are methods comprising DS / PAGE and protein staining or Western blotting, followed by quantitative analysis, eg concentration scanning of the resulting gel or blot. A specific increase in the amount of a recombinant protein, polypeptide or peptide relative to the amount in a natural cell is the relative abundance of a particular polypeptide with respect to other proteins produced by the host cell;
For example, over-expression is visible on the gel.

The expression vectors provided herein contain a polynucleotide encoding a modified DT or fusion protein thereof in a form suitable for expression of the polynucleotide in a host cell. Expression vectors are generally selected based on the host cell to be used for expression and have one or more regulatory sequences operably linked to the polynucleotide sequence to be expressed.
One skilled in the art will recognize that it can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. The expression vectors described herein are introduced into a host cell to produce a protein, such as a fusion protein, encoded by a polynucleotide (such as a modified DT or DT fusion protein) as described herein. be able to.

Expression vectors can be designed for the expression of modified DT or DT fusion proteins in prokaryotic or eukaryotic cells. The presence of a single DT gene inside a eukaryotic cell will kill the cell. Specifically, the toxin binds to EF-tu that is required for translation and ribosylation. Thus, DT can only be expressed in eukaryotic cells with modified EF-tu that are no longer recognized by DT (eg, Liu et al., Pro
tein Expr. Purif. , 30: 262-274 (2003); Phan e
t al. , J .; Biol. Chem. 268 (12): 8665-8 (1993);
Chen et al. Mol. Cell Biol. , 5 (12): 3357-60
(1985); Kohne et al. , SomaT Cell Mol. Genet
. 11 (5): 421-31 (1985); Moehring et al. , Mol
. Cell Biol. 4 (4): 642-50 (1984)). Modified DT
Alternatively, the fusion protein can be expressed in bacterial cells such as E. coli (Bis
hai et al. , J .; Bacteriol, 169 (11): 5140-51 (1
987)). The type of expression and activity and the level of host protease expression are considered and depend on the cleavage site present in the engineered DT poison. The unique expression host protease expression profile will adversely affect the yield of DT fusion toxin produced (Bishai)
et al. , Supra (1987)). To the extent that the cell selectivity of fusion proteins obtained by altering this essential cleavage site can be regulated, it is envisioned that such cleavage site mutants may be VLS-modified poisons (Gordon). et al.,
Infect Immun. 63 (1): 82-7 (1995); Gordon et
al. , Infect Immun. 62 (2): 333-40 (1994); Va
llera et al. , J .; Natl. Cancer Inst. 94: 597-
606 (2002); Abi-Habib et al. , Blood. 104 (7)
: 2143-8 (2004)). Alternatively, the expression vector can be transcribed and translated in vitro.

The application further provides gene delivery vehicles for delivering polynucleotides to cells, tissues, mammals for expression. For example, the polynucleotide sequences of the present invention can be administered to a gene delivery vehicle locally or systemically. These structures can utilize viral or non-viral vector approaches in an in vitro or in vitro manner. Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be constitutive or regulatory.
The invention encompasses gene delivery vehicles capable of expressing the intended polynucleotide, including viral vectors. For example, Q developed a system using PG13 packing cells.
have created recombinant retroviruses with DT fragments that provide a method of killing cancer cells and using DT as an element of a suicide vector. Qiao et al. , J
. Virol. 76 (14): 7343-8 (2002).

Expressed DT mutants and DT fusion proteins can be tested for their functional activity. Methods for testing DT activity are well known in the art. For example,
The VLS effect of DT mutants and DT fusion proteins was determined as described in Example 2.
Can be tested with UVEC. The ribosyltransferase activity of DT variants and DT fusion proteins can be tested by the ribosyltransferase assay described in Example 3. The cytotoxicity of DT variants and DT fusion proteins is shown in Example 4.
Can be tested as described in.

The application also provides a purified polypeptide that is expressed using one or more methods described herein, and in a preferred embodiment provides a substantially purified polypeptide. To do. The term “purified” as used herein is a proteinaceous composition that can be isolated from a mammalian cell or recombinant host cell, wherein at least one polypeptide can be obtained in nature. It is intended to refer to a proteinaceous composition that has been purified to any degree as compared to the state, ie its purity in the cell extract. Thus, a purified polypeptide also refers to a wild-type or modified polypeptide that does not exist in the environment in which it naturally occurs.

When the term “substantially purified” is used, this means that a particular polypeptide forms the major component of the composition, eg, about 50% or more of the protein in the composition. Will be referred to. In one embodiment, the substantially purified polypeptide is about 60%, about 70%, about 80%, about 90%, about 95%, about 9% in the composition.
It will constitute more than 9% or even more polypeptide.

As applied to the present invention, a polypeptide that is “purified to homogeneity” means that the polypeptide has a purity level that is substantially free of other proteins and biological components. . For example, a purified polypeptide will often not contain enough other protein components so that degradation sequencing can be performed.

Various methods for quantifying the degree of purification of the polypeptide will be known to those of skill in the art in light of the disclosure herein. These methods include, for example, measuring the specific protein activity of the fraction, or evaluating the number of polypeptides in the fraction by gel electrophoresis.

In order to purify the desired polypeptide, natural or recombinant compositions containing at least some specific polypeptides will be subjected to fractionation to remove various other components from the composition. . In addition to the techniques described in detail herein below, various other techniques suitable for use in protein purification will be well known to those skilled in the art. These techniques include, for example, precipitation or thermal denaturation using ammonium sulfate, PEG, antibodies, etc., followed by centrifugation, chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography. Graphic processes; equipotential point electrophoresis, gel electrophoresis; and combinations of these and other techniques.

Another example is the purification of a specific fusion protein using a specific binding partner. Such purification methods are routine in the art. The present invention relates to DNA for specific proteins.
Any fusion protein purification method can now be performed since it provides the sequence. This may involve the production of specific protein-glutathione S-transferase fusion proteins, expression in E. coli and isolation to homogeneity using affinity chromatography on glutathione-agarose or poly on the N- and C-termini of proteins. Illustrated by the generation of histidine label as well as subsequent purification using Ni-affinity chromatography.
However, a given number of DNAs and proteins are known or can be identified and amplified using the methods described herein, and any purification method can be used here.

It is not a general requirement that the polypeptide always be provided in its most produced state. Indeed, substantially less purified polypeptides that are nevertheless abundant in the desired protein composition compared to their native state are intended to have utility in certain embodiments. . A polypeptide that exhibits a low degree of relative purification may have advantages in all recovery of the protein product or in maintaining the activity of the expressed protein.

A method for preparing a composition comprising (a) a polypeptide having the amino acid sequence of SEQ ID NO: 4-147 or a polynucleotide encoding a polypeptide having two or more such modifications Provided herein is a method comprising: (b) expressing the polypeptide in a host cell containing the vector. In one embodiment, the composition is produced in such a way that the composition has reduced binding to human vascular endothelial cells (HUVEC) compared to a DT molecule having the sequence of SEQ ID NO: 2 or 200 Has activity. Also, a method of preparing a composition comprising: (a) assembling a vector containing a polynucleotide encoding a toxin having at least one T cell epitope modification; (b) containing the polypeptide and the vector Provided herein is a method comprising expressing in a host cell. In one embodiment, the composition is manufactured in such a manner and the toxin is DTT. In one embodiment, the composition is produced in such a way, said composition having a reduced immunogenicity compared to a DT molecule having the sequence of SEQ ID NO: 2 or 200.

Provided herein are methods for preparing modified toxins having reduced immunogenicity compared to unmodified toxins. The method comprises (a) vector assembly containing a nucleic acid sequence encoding a toxin, wherein the modified diphtheria toxin has an amino acid sequence as shown in SEQ ID NO: 2 having one or more amino acid modifications therein Consisting of diphtheria toxin, in which case at least one amino acid modification is made within the T cell epitope, and in this case said modified diphtheria toxin has cytotoxicity comparable to unmodified diphtheria toxin, and (b ) Comprising the step of expressing said polypeptide in a host cell containing said vector.

Another embodiment includes a method of preparing a modified diphtheria toxin having reduced immunogenicity and reduced binding activity to human vascular endothelial cells (HUVEC) compared to unmodified diphtheria toxin. The method comprises (a) assembling a vector containing a nucleic acid sequence encoding a modified diphtheria toxin, wherein the modified diphtheria toxin has one or more amino acid modifications therein as shown in SEQ ID NO: 2 or 200 Wherein at least one amino acid modification is within a T cell epitope, SEQ ID NO:
Performed in a region selected from the group consisting of 2 or 200 residues 7-9, 29-31 and 290-292, within a (x) D (y) motif, within a B cell epitope, or a combination thereof, And in this case, said modified diphtheria toxin has cytotoxicity comparable to unmodified diphtheria toxin, and (b) comprises the step of expressing said polypeptide in a host cell containing said vector. In one non-limiting example, a modified VLS (x) D / E (
y) The motifs are A, S, E, F, C, M, T, W, Y, P, H, Q, D, N, K, R,
V or I by amino acid residues selected from G, L and the modified or unusual amino acids of Table 1
Modification at position (x), and in this case the modification of position D / E is A, S, E, I, V, L, F, C, M, G, T , W, Y, P, H, Q, N, K, R and the modifications in Table 1 or substitution of D / E with amino acid residues selected from among the unusual amino acids. In one embodiment, the modification at position (y) is I, F, C, M, A, G, T, W,
Y, P, H, E, Q, D, N, K, R, S, L, V and Table 1 modification or substitution with abnormal amino acids.

The unmodified diphtheria toxin can have, for example, the amino acid sequence of SEQ ID NO: 2 or 200, or any one amino acid sequence of SEQ ID NO: 4-147.

Bacterial and plant complete toxins often contain two disulfide-linked chains, namely the A chain and the B chain. The B chain has both a cell binding region (its receptor is often unspecified) and a translocation region, facilitating the insertion of the A chain into the cytosol through the membrane of the acidic cell compartment. . The A chain then kills the cell after uptake. For its in vivo use, the ligand and toxin are linked in such a way as to remain stable while passing through the bloodstream and tissues, but not in the target cell so that the toxin moiety can be released into the cytosol. It is stable. However, from a pharmacological point of view, it may be desirable to use the smallest molecule that can nevertheless provide an appropriate biological response. Therefore,
It may be desirable to use smaller A chain peptides that will provide an adequate anti-cellular response. To achieve this goal, DT can be “cleaved” and still retain the proper toxin activity. If desired, it is proposed that this cleaved A chain can be used in a fusion protein according to the embodiments described herein.

Alternatively, it can be found that application of recombinant DNA technology to the toxin moiety can provide additional advantages. In view of the fact that biologically active DT is currently cloned and recombinantly expressed,
Nonetheless, smaller variant peptides or variant peptides that exhibit appropriate toxin activity can be identified and prepared. Also, the fact that DT is currently cloned allows for the application of site-directed mutagenesis, whereby DT A chain, toxin-derived peptides can be easily prepared and screened and is described herein. Another useful moiety can be obtained for use with the described compounds. Once identified, these portions exhibit reduced ability to promote VLS, EC damaging activity and / or other effects of such sequences described herein or known to those of skill in the art. Can be mutated to create a toxin.

A fusion protein comprising a modified diphtheria toxin and a non-diphtheria toxin polypeptide prepared by such a method, wherein the non-diphtheria toxin polypeptide is an antibody or an antigen-binding fragment thereof, EGF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, I
L-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL
-14, IL-15, INFα, INFγ, GM-CSF, G-CSF, M-CSF, T
Provided herein are fusion proteins that are selected from NF, VEGF, ephrin, BFGF, TGF and their cell binding moieties. In one embodiment, the non-diphtheria toxin polypeptide is, for example, IL-2 or a cell binding portion thereof.
In certain embodiments, the fusion protein or toxin further contains at least another substance. Such a substance can be a molecule or moiety, such as, but not limited to, at least one effector (therapeutic drug moiety) or reporter molecule (as described elsewhere herein).
Detectable label).

IX. Detectable Labels The present invention provides fusion proteins to target epitopes, such as epitopes expressed in diseased tissues or cells that cause disease (eg, IL-2 receptors on the surface of cancer cells).
In certain embodiments, the fusion protein contains a modified DT as described herein. In another embodiment, the fusion protein further contains a second substance. Such a substance can be, for example, a molecule or moiety such as a reporter molecule or a detectable label. A reporter molecule is any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to polypeptides include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, An example is biotin. As a detectable label, its specific functional properties,
And / or compounds and / or elements that can be detected by chemical properties, the use of which allows them to detect and / or quantitate if desired the bound polypeptides. A number of suitable detectable (contrast) materials are known in the art since there are ways to attach them to polypeptides (eg, US Pat. No. 5,0
21,236, 4,938,948 and 4,472,509, each of which is incorporated herein by reference). The contrast moiety used can be a paramagnetic ion, a radioisotope, a fluorescent dye, an NMR detectable substance, an X-ray contrast moiety.
Molecules containing azide groups can also be used to form covalent bonds to proteins through reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter & Haley
1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-specific optical probes to identify nucleotides that bind proteins in crude cell extracts (Owens & Haley, 1987; Atherton et a
l. , 1985). 2- and 8-azido nucleotides have also been used to locate the nucleotide binding domain of purified proteins (Khaton et al.
, 1989; King et al. 1989, and Dholakia et al.
1989), and can be used as a polypeptide binding agent.

X. Compositions and therapeutic uses Each of the compounds described herein can be used as a composition when combined with an acceptable carrier or excipient. Such compositions are useful for in vitro analysis or for administration to a subject in vivo or in vitro to treat the subject with the disclosed compounds.

Thus, in addition to the active ingredient, the pharmaceutical composition can contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other substances well known to those skilled in the art. Such substances are
It should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration.

A pharmaceutical formulation containing a protein of interest, eg, an antibody, identified by the methods described herein can be used to produce a protein having a desired degree of purification in any physiologically acceptable carrier, excipient or stabilizer ( Remington's Pharmaceutical Science
es 16th edition, Osol, A.E. Ed. (1980)) can be prepared for storage in the form of a lyophilized formulation or an aqueous solution. An acceptable carrier,
Excipients or stabilizers are not toxic to the recipient at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants such as Preservatives (eg, octadecyldimethylbenzylammonium chloride; hexamesonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; Cyclohexanol; 3
-Pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, glutamine, asparagine Histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium Metal complexes (eg Zn-protein complexes); and / or nonionic surfactants, eg T
WEEN (registered trademark), PLURONICS (registered trademark) or polyethylene glycol (
PEG).

The formulations described herein can also contain more than one active compound as required for the particular indication being treated. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

An acceptable carrier is physiologically acceptable to the patient being administered and retains the therapeutic properties of the compound administered therewith / in it. Acceptable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences (
18th Edition, ed. A. Gennaro, Mack Publishing
g Co. , Easton, PA 1990). One typical carrier is physiological saline. As used herein, the phrase “pharmaceutically acceptable carrier” carries the compound of interest from the site of administration of one organ or part of the body to another organ or part of the body, or in an in vitro assay system, or It means a pharmaceutically acceptable substance, composition or vehicle involved in transport, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the administered subject. An acceptable carrier should not alter the specific activity of the compound of interest. Typical carriers and excipients are provided elsewhere herein.

In one embodiment, a pharmaceutically acceptable or physiologically containing solvent (aqueous or non-aqueous), solution, emulsion, dispersion medium, coating, isotonic and absorption enhancer or retarder that is compatible with pharmaceutical administration An acceptable composition is provided herein. Accordingly, a pharmaceutical composition or pharmaceutical preparation refers to a composition suitable for pharmaceutical use in a subject. Pharmaceutical compositions and formulations contain an amount of a compound described herein, eg, an effective amount of a modified fusion protein described herein, and a pharmaceutically or physiologically acceptable carrier.

The composition can be formulated to be compatible with a particular route of administration (systemic or local). Thus, the compositions contain a carrier, diluent or excipient suitable for administration by various routes. Pharmaceutical compositions for oral administration can be in the form of tablets, capsules, powders or solutions.
A tablet may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Saline solutions, dextrose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

In another embodiment, the composition further contains an acceptable additive, as necessary, to improve the stability of the compound in the composition and / or to control the release rate of the composition. can do. Acceptable additives do not change the specific activity of the target compound. Typical acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose,
Examples include galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Acceptable additives can be combined with acceptable carriers and / or excipients such as dextrose. Alternatively, typical acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase peptide stability and reduce solution gelation. Surfactants can be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition during storage.

The pharmaceutical composition can be administered subcutaneously, intramuscularly, intraperitoneally, orally or intravenously. Aerosol delivery of the composition is also contemplated herein using conventional methods.
For example, intravenous delivery is currently possible by cannula or direct injection or by an ultrasound guided fine needle. Mishra (Mishra et al., Expert O
pin. Biol. 3 (7): 1173-1180 (2003)) provides intratumoral injection.

Preparations for enteral (oral) administration include tablets (coated or uncoated), capsules (hard or soft),
It can be contained in microspheres, emulsions, powders, granules, crystals, suspensions, syrups or elixirs. Conventional non-toxic solid carriers such as the pharmaceutical great mannitol,
Lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate) can be used to prepare solid formulations. Supplementary active compounds (eg, preservatives, antibacterials, antivirals and antifungals) can also be included in the formulation. Liquid formulations can also be used for enteral administration. The carrier can be a variety of oils such as petroleum, animal oils, vegetable oils or synthetic oils such as peanut oil, soybean oil,
It can be selected from mineral oil and sesame oil. Suitable pharmaceutical excipients include, for example, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate,
Examples include glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.

Injectable compositions include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include
Saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippa
ny, N.M. J. et al. ) Or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Flowability can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Examples of the antibacterial agent and antifungal agent include paraben, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example sugars,
Polyalcohols such as mannitol, sorbitol and sodium chloride may be included in the composition. The resulting solution can be packaged for use as is or lyophilized. The lyophilized formulation can later be combined with a sterile solution prior to administration.

The composition can be conventionally administered, eg, intravenously, eg, by unit dose injection.
For injection, the active ingredient may be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has appropriate pH, isotonicity and stability. For example,
Appropriate solutions can be prepared using isotonic vehicles such as sodium chloride solution, Ringer's solution, and lactated Ringer's solution. If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives may be contained.

In one embodiment, the composition is lyophilized. Where the composition is contemplated for use in medicine or in any of the various methods provided herein, the composition is
It is contemplated that the composition is substantially free of pyrogens so as not to cause an inflammatory response or an unsafe allergic reaction.

Acceptable carriers can contain compounds that stabilize, enhance or retard absorption or clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose or dextrin; low molecular weight proteins; compositions that inhibit peptide clearance or hydrolysis; excipients or other stabilizers and / or buffers. .
Examples of substances that delay absorption include aluminum monostearate and gelatin. Detergents can also be used to stabilize, increase or decrease the absorption of pharmaceutical compositions containing liposomal carriers. To protect against digestion, the compound can be complexed with the composition to make it resistant to acid and enzymatic hydrolysis, or the compound can be complexed in a moderately resistant carrier such as a liposome. it can. Means of protecting compounds from digestion are known in the art (eg, Fix (1996) Pharm. Res., 13: 1760-176, which describes lipid compositions for oral delivery of therapeutic agents).
4; Samenen (1996) J. MoI. Pharm. Pharmacol. 48: 119
-135 and U.S. Pat. No. 5,391,377).

For intravenous injections or injections at difficult sites, the active ingredient is in the form of a parenterally acceptable aqueous solution that does not contain pyrogens and has appropriate pH, isotonicity and stability. is there. One skilled in the art can adequately prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride solution, Ringer's solution, and lactated Ringer's solution. Preservatives, if necessary
Stabilizers, buffers, antioxidants and / or other additives may be included.

The phrase “pharmaceutically acceptable” refers to physiologically acceptable and typically allergic side effects or similar adverse side effects such as increased acute gastric peristalsis, dizziness, etc. when administered to humans. Refers to molecular entities and compositions that do not occur.

The term “unit dose” when used in reference to a therapeutic composition refers to a physically discrete unit suitable as a unit dosage form for humans, each unit comprising the required diluent, ie carrier or Contains a predetermined amount of active substance calculated to produce the desired therapeutic effect in conjunction with the vehicle.

The composition can be administered in a manner compatible with the formulation and in a therapeutically effective amount. The amount administered will depend on the subject being treated, the ability of the subject's immune system to utilize the active ingredients and the degree of binding ability desired. The exact amount of active ingredient required for administration depends on the judgment of the practitioner and is specific to each individual. Treatment regimes suitable for initial administration and booster immunization can be varied, but are represented by repeated administrations at one or more time intervals until the first administration followed by the next injection or other administration. Or, in many cases, 10 nanomolar to 1 in the blood
Sufficient continuous intravenous infusion to maintain a concentration of 0 micromolar is contemplated.

A physician or veterinarian can readily determine and prescribe the “effective amount” (ED ₅₀ ) of the required composition. For example, a physician or veterinarian can start with a dose of the compound used in the composition that is less than that required to achieve the desired therapeutic effect, and formulate the formulation until the desired effect is achieved. It can be gradually increased.

As used herein, a “therapeutically effective amount” is an amount that at least partially achieves a desired therapeutic or prophylactic effect in an organ or tissue. In one example, the amount of modified DT necessary to achieve a prophylactic and / or therapeutic treatment of a disease is not itself fixed. Modified D to be administered
The amount of T-fusion toxin varies with the type of disease, the severity of the disease and the size of the mammalian species suffering from the disease. In general, the amount is in the range used for other cytotoxic drugs used in the treatment of cancer, but in some cases due to the specificity and increased toxicity of the modified DT fusion toxin, Lesser amounts will be required. Attempts to deliver enhanced locally increased amounts of fusion toxins to specific sites, as can be achieved in certain situations and with currently available techniques such as (cannula or convection enhanced delivery, selective release) also May be desirable (L
ask et al. , J .; Neurosurg. 87: 586-5941 (997).
Laske et al. , Nature Medicine, 3: 1362-13.
68 (1997), Rand et al. , Clin. Cancer Res. , 6:
2157-2165 (2000); Engebraaten et al. , J .; Can
cer, 97: 846-852 (2002), Prados et al. , Proc.
ASCO, 21: 69b (2002), Pickering et al. , J .; CLi
n. Invest. 91 (2): 724-9 (1993)).

One embodiment contemplates the use of a composition described herein to prepare a medicament for treating a condition, disease or disorder described herein. Medications are for patients /
It can be formulated based on the physical properties of the subject and can be formulated into single or multiple formulations depending on the stage of the condition, disease or disorder. The medication can be packaged in an appropriate package with an appropriate label for distribution to hospitals and clinics, where the label is for indications about the treatment of a subject having the disease described herein. belongs to. The medicament can be packaged as a single or multiple units. Instructions for administration of the formulations and compositions can be included in the package as described below.

The invention further relates to a pharmaceutical composition comprising the modified toxin described above or a fusion protein thereof and a pharmaceutically acceptable carrier.

Sterile injectable solutions can be prepared by mixing a predetermined amount of the active ingredient in a suitable solvent, optionally with one or a combination of the ingredients listed above, and then sterile filtering.
Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization, which is a pre-sterilized filtration of the active ingredient and any additional desired ingredients powder. From the solution.

In certain embodiments, this mixture is further purified to obtain a formulation that essentially contains the fusion protein. This purification can be achieved by further chromatographic separation,
For example, it can be accomplished by affinity chromatography using a salt gradient to elute various species of immunotoxin and gel filtration that separates the immunotoxin from large molecules.

The gel used for purification of the compounds described herein is a three-dimensional network having a random structure. Molecular sieve gels are cross-linked polymers that do not bind or react with the material to be analyzed or separated. For gel filtration applications, gel materials are generally not charged. The space within the gel is filled with liquid and the liquid phase constitutes the majority of the gel volume. Substances commonly used in gel filtration columns include dextran, agarose and polyacrylamide.

Dextran is a polysaccharide composed of glucose residues, and SEPHADEX (Ph
amacia Fine Chemicals, Inc. ). The beads are prepared with various degrees of cross-linking with the aim of separating different sized molecules by providing various pore sizes. Alkyldextran is N, N'-
Cross-linked with methylene bisacrylamide and SEPHACRYL-S100-S10
00, which makes it possible to prepare powerful beads that sort over a wider range than if SEPHADEX can be achieved.

Polyacrylamide can also be used as a gel filtration medium. Polyacrylamide is a polymer of crosslinked acrylamide prepared using N, N′-methylenebisacrylamide as a crosslinking agent. Polyacrylamide is available in various pore sizes from Bio-Rad Laboratories (USA) for use in the separation of particles of various sizes.

Gel materials swell in water and several organic solvents. Swelling is a process in which pores are filled with a liquid that is used as an eluent. As smaller molecules enter the pores, these advancements through the gel are delayed compared to larger molecules that do not enter the pores and form the basis for separation. Beads are available in various finenesses used in various applications. The coarser the beads, the faster the flow and the worse the separation. Ultra fine grades can be used for maximum separation, but flow is very slow. The fine grade is used for preparation operations on large columns that require faster flow rates. Coarse grades are for large formulations where separation is less important than time, or for separation of molecules that have a large difference in molecular weight.

Affinity chromatography is generally based on the recognition of proteins by substances such as ligands or antibodies. The column material can be synthesized by covalently linking a binding molecule, such as an activated dye, for example, to an insoluble matrix. The column material then allows the desired substance to be adsorbed from the solution. Next, the condition is changed to a condition in which binding does not occur, and the substrate is eluted. The prerequisites for successful affinity chromatography are that the matrix must adsorb molecules, that the ligands must be linked without changing their binding activity, and that the ligands are sufficiently robust in their binding. And must be able to elute the material without destroying it.

One embodiment of the compounds described herein is an affinity chromatography method wherein the matrix is a reactive dye-agarose matrix. Blue-SEPHA
ROSE (Cibacron Blue 3GA and agarose or SEPHAROSE
Can be used as an affinity chromatography matrix. Moreover, SEPHAROSE CL-6B is Sigma Che
Available as Reactive Blue 2 from the Mic Company. This matrix allows the fusion proteins to be separated by directly binding the fusion proteins and eluting with a salt gradient.

Compositions containing the modified toxins are provided herein. In one embodiment, the modified toxin consists of diphtheria toxin, said modified diphtheria toxin containing an amino acid sequence as set forth in SEQ ID NO: 2 or 200 having one or more amino acid modifications therein, At least one amino acid modification may be within a T cell epitope, or SEQ ID NO: 2 or 20
(X) D / E of a region selected from among residues 0-9, 29-31 and 290-292 of 0
(Y) performed within a motif, within a B cell epitope, or a combination thereof, and the modified diphtheria toxin has cytotoxicity comparable to unmodified diphtheria toxin. In one non-limiting example, the modified VLS (x) D / E (y) motif is A, S, E, F, C
, M, T, W, Y, P, H, Q, D, N, K, R, G, L, and substitution of V or I with amino acid residues selected from the modifications or abnormal amino acids in Table 1 Contains a modification at position (x), and modifications at position D / E are A, S, E, I, V, L, F, C, M, G, T, W,
Y, P, H, Q, N, K, R and the modification of Table 1 or substitution of D or E with an amino acid residue selected from among the abnormal amino acids. In one embodiment, the modification at position (y) is
I, F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K, R, S, L, V,
And Table 1 modification or substitution with unusual amino acids.

The unmodified diphtheria toxin can have, for example, the amino acid sequence of SEQ ID NO: 2 or 200 or the amino acid sequence of any one of SEQ ID NO: 4-147.

The composition contains a modified diphtheria toxin, which composition has reduced immunogenicity, reduced binding activity to human vascular endothelial cells (HUVEC) and combinations thereof compared to unmodified diphtheria toxin. Such compositions include, but are not limited to, antibodies or antigen binding fragments thereof, EGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6.
, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, INFα, INFγ, GM-CSF, G-CSF, M-CSF
Further comprising a non-diphtheria toxin polypeptide containing TNF, VEGF, ephrin, BFGF and TGF. A non-diphtheria toxin polypeptide can also be a fragment of such a polypeptide, eg, a cell binding portion thereof. In one embodiment, the non-diphtheria toxin polypeptide is IL-2 or a cell binding portion thereof.

Modified toxins and their fusion proteins that have reduced immunogenicity, reduced binding to HUVEC, or a combination thereof and at the same time maintain cytotoxicity are useful for various lymphoid malignancies (eg, cancer), solid tumors and It can be used to treat non-malignant diseases such as GVHD or psoriasis.

In an exemplary embodiment, the T cell epitope-modified DT fusion toxin of the present invention is used in medical disorders such as cancers such as T cell lymphoma, or unwanted cells to which a target ligand can selectively bind. It is administered to a subject such as a mammal (eg, human) suffering from a non-malignant disease characterized by the presence of the species.

Denileukin diftitox has been shown to be effective in treating subjects with previously treated cutaneous T cell lymphomas (Chin and Foss (
2006) Clinical Lymphoma and Myeloma, 7 (3):
199-204; Talpur et al. (2006) J. Am. Investigat
ive Dermatology, 126: 575-583). Briefly, Denileukin diftitox is intravenously administered for 3 or 5 consecutive days, 4 μg / kg / day, 9 μg / k.
It was administered for 3-21 cycles at a dose of g / day or 18 μg / kg / day. A total reaction rate of 51% was observed. Denileukin diftitox is approved by the US Food and Drug Administration in the United States for the treatment of cutaneous T-cell lymphoma.

Provided herein is a method of treating a subject having cutaneous T cell lymphoma by administering a modified DT fusion protein described herein.

Denileukin diftitox has been shown to be effective in treating subjects with relapsed / refractory T-cell and B-cell non-Hodgkin's lymphoma (Dang et
al. , (2006) Br. J. et al. Haematology, 136: 439-447;
Dang et al. , (2004) J. Am. Clin. Oncol. , 22: 4095-
4102). Briefly, eligible patients were denyleukin diftitox (18 μg / kg
/ Day) every 3 weeks for 5 days for a maximum of 8 cycles. Such a treatment regimen was well tolerated by patients and was effective in treating relapsed / refractory T-cell and B-cell non-Hodgkin lymphoma.

Relapsed / refractory T by administering a modified DT fusion protein described herein
Provided herein are methods for treating a subject having a cellular or B-cell non-Hodgkin lymphoma.

In a phase II clinical trial, ONTAK® in combination with rituximab is
It has been shown to be significantly effective in treating patients with relapsed / refractory B-cell non-Hodgkin lymphoma. Accordingly, provided herein are methods for treating a subject having relapsed / refractory B-cell non-Hodgkin's lymphoma by administering a modified DT fusion protein described herein.

Denileukin diftitox has been shown to be effective in treating subjects with subcutaneous adipose tissue-like T-cell lymphoma (McGinnis et al., (2
002) Arch. Dermatol. 138: 740-742). Briefly, female patients previously treated with bexarotene and interferon alpha relapsed within 2 months of treatment. This patient was then treated with intravenous Denileukin diftitox (9 μg / kg /
One cycle of treatment for 5 days). Clinical remission was observed with resolution of all skin diseases and systemic symptoms were obtained two weeks after completion of the third cycle of Denileukin diftitox.

Provided herein are methods for treating a subject having subcutaneous adipose tissue-like T-cell lymphoma by administering a modified DT fusion protein described herein. In one non-limiting embodiment, if necessary, the subject is treated in combination with one or more other therapies, such as bexarotene and / or interferon alpha.

Denileukin diftitox has been shown to be effective in treating subjects with extranodal natural killer / T cell lymphoma, nasal type (Kerr et al.
. , (2006) Br. J. et al. Dermatology, 154: 988-991). Briefly, the rapidly progressive Epstein-Barr virus positive nasal extranodal natural killer / T
A 58-year-old man with cellular lymphoma was treated with a combination of bexarotene and denileukin diftitox. Significant regression of skin tumors was observed after the first cycle of Denileukin diftitox and 5 in the monthly cycle of Denileukin diftitox.
Maintained for months.

Provided herein is a method of treating a subject with extranodal natural killer / T cell lymphoma, nasal form, by administering a modified DT fusion protein described herein in combination with bexarotene.

Denileukin diftitox has been shown to be effective in treating subjects with previously treated chronic lymphocytic leukemia (Frankel et al.
(2006) Cancer, 106 (10): 2158-2164). Simply put,
Denileukin diftitox is 18 μg every 21 days for 60 minutes as an intravenous infusion.
A maximum of 8 cycles were administered at a dose of / kg / day. Overall, patients showed a decrease in peripheral chronic lymphocytic leukemia (CLL) cells, a reduction in lymph node size, and in some cases a remission confirmed over time from a bone marrow biopsy. In some cases, treated patients were chemoresistant to fludarabine (Morgan et al., (2004) Clin.
. Cancer Res. , 9 (10 Pt 1): 3555-3561).

Provided herein is a method of treating a subject having chronic lymphocytic leukemia by administering a modified DT fusion protein described herein.

Denileukin diftitox has been shown to be effective in treating subjects with human T cell lymphocyte-directed virus 1 associated acute T cell leukemia / lymphoma (
Venuti et al. , (2003) Clin. Lymphoma, 4 (3): 1
76-180). Simply put, four cycles of Denileukin diftitox are administered,
This resulted in restoration of standard blood pressure and reduction of myelofibrosis. After disease progression, 4 cycles of hyper-CVAD (multi-particulate cyclophosphamide / doxorubicin / vincristine / decadrone) were administered to achieve complete clinical remission. The patient received maintenance therapy with Denileukin diftitox for 1 year.

A method of treating a subject having human T cell lymphocyte-directed virus 1 associated acute T cell leukemia / lymphoma by administering a modified DT fusion protein described herein in combination with hyper CVAD therapy is provided herein. Provided in

Denileukin diftitox has been shown to be effective in treating subjects with solid tumors (Eklund and Kuzel. Expert Rev.
Anticancer Ther. 2005 Feb; 5 (l): 33-8). Accordingly, provided herein are methods for treating a subject having one or more solid tumors by administering a modified DT fusion protein described herein. Typical solid tumors include, but are not limited to, skin, melanoma, lung, pancreas, breast, ovary, colon,
Rectum, stomach, thyroid, larynx, ovary, prostate, colorectal, head, neck, eye, mouth, pharynx, esophagus,
Tissue, organ solids selected from breast, bone, testis, lymph, bone marrow, bone, sarcoma, kidney, sweat gland, liver, kidney, brain, gastrointestinal tract, nasopharynx, urogenital tract, muscle and other tissues A tumor.

Acute graft-versus-host disease (aGVHD) is mediated in part by activated T cells that express a high affinity receptor for IL-2 that is recognized by Denileukin diftitox. In a phase II study of patients with steroid resistant aGVHD,
5 μg / kg was treated once a day, 1-5 days, then once a week, on the 8th, 15th, 22nd and 29th days of the study. Another group of patients was treated with 9 μg on the same dosing schedule as above.
Treated with / kg usage. Responses were evaluated on days 36 and 100. 41% of patients respond, all respond in full on day 36, and 27% respond in day 100 (4 complete responses and 2 partial responses).

Provided herein is a method of treating a subject having aGVHD by administering a modified DT fusion protein described herein.

Psoriasis is an immune-mediated skin disease in which T cells are chronically stimulated by skin antigen-presenting cells. Psoriasis is a chronic recurrent disease that requires intermittent treatment. Denileukin diftitox was shown to effectively target activated T cells and reversed psoriasis. However, the side effect of treatment was vascular leakage syndrome (Walsh and Shear. (2
004) CMAJ, 170 (13): 1933-1941). In a Phase II study of patients with severe psoriasis, 35 patients were treated with three doses of ONTAK® (0.5
, 1.5 or 5 μg / kg / day) and received 3 doses per week for 8 weeks. 8 of 15 patients (treated with 5 or 1.5 μg / kg / day)
Psoriasis Area and Severity Index (Psoriasis Area and Severity)
Index (PASI) and Physicin 'Global A
sessment) (PGA) showed more than 50% reduction in symptoms.
Four patients (all who received treatment at a dose of 5 μg / kg / day) benefited from a two-stage pathology improvement on a five-stage PGA scale.

Provided herein are methods of treating a subject having psoriasis by administering a modified DT fusion protein described herein.

Also contemplated herein are methods that provide maintenance therapy by administering a non-immunogenic DT fusion protein described herein.

Dannull et al. (J. Clin. Invest., 115 (12): 3623-36.
33 (2005)), immunization with RNA-transfected dendritic cells (DC) is an effective method of stimulating strong T cell responses in patients with metastatic cancer (Su). et al., 2003. Cancer Res., 63: 3127-21
33; Heiser et al. , 2002. J. et al. Clin. Invest. 109
: 409-417). CD4 + T cells that constitutively express the IL-2 receptor α chain (CD25
) Function in regulatory capacity by inhibiting the activation and function of other T cells (Shebach, EM 2001. J. Exp. Med., 193: F41-F46).
). Their physiological role is to protect the host from the development of autoimmunity by modulating the immune response to antigens expressed by normal tissues (Jonuleite et al.
t al. 2000. J. et al. Exp. Med. 192: 1213-1222; Read
and Powerrie. 2001. Curr. Opin. Immunol. , 13: 6
44-649). Since tumor antigens are mostly self-antigens, T regulatory cells (Tregs)
Can also prevent a tumor-bearing host from initiating an effective anti-tumor immune response. Previous studies have shown that an increased number of CD4 + CD25 + Treg can be seen in patients with advanced cancer (Woo et al., 2002. J. Immunol., 168: 4272).
-4276) and high Treg frequency is associated with decreased survival (Curiel et
al. , 2004. Nat. Med. 10: 942-949).
The important role of CD4 + CD25 + Treg in controlling tumor growth was further emphasized by demonstrating that lack of Trag using anti-CD25 antibodies can elicit effective anti-tumor immunity in mice (Shimizu et al., 1999. J. Im
munol. 163: 5211-5218; Onizuka et al. , 1999
. Cancer Res. 59: 3128-3133). In addition, anti-CD25 therapy is G
Increased therapeutic efficacy of M-CSF secreting B16 tumor cells and prolonged survival of animals with tumors (Summuller et al., 2001. J. Exp. Med., 194: 82).
3-832). Cumulatively, these experimental data may increase the effectiveness of cancer treatment by administration of agents that cause preferential depletion of CD4 + CD25 + Treg, eg, compounds that target cells expressing the IL-2 receptor CD25 subunit. It suggests that you can.

Recombinant IL-2 diphtheria toxin is used to eliminate CD25-expressing Treg in patients with metastatic renal cell carcinoma (RCC), DAB389IL-2 (denileukin diftitox and O
NTAK® (also known as NTAK®). DAB389I as a result of binding to the high affinity IL-2 receptor, subsequent internalization, and enzyme inhibition of protein synthesis.
The cytotoxic effect of L-2 occurs and eventually leads to cell death.

DAB389IL-2 is an intermediate or low level expression of CD25 with other PBMC or CD4
+ Human PBMC in a dose-dependent manner without apparent bystander toxicity to T cells
Was demonstrated to selectively exclude Tregs. Treg deficiency resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro, but DAB389IL-2 was used prior to the T cell priming phase and was removed during T cell priming. Was only the case. The lack of Tregs in RCC patients by immunization with DAB389IL-2 followed by tumor RNA-transfected DCs resulted in improved stimulation of tumor-specific T cells compared to administration of tumor RNA-transfected DC alone. CD4 + CD25 high Tregs can be eliminated using a single dose of DAB389IL-2 without apparent bystander toxicity and without affecting the function of other cells expressing CD25. DAB389IL-2 significantly reduced the number of Tregs present in the peripheral blood of RCC patients, decreased the amount of FoxP3 transcript derived from peripheral blood, and suppressed Treg-mediated immunosuppressive activity in vivo. Also, the significantly higher frequency of tumor-specific CD8 + T cells when compared to subjects receiving DC alone is the combined DAB389I.
It can be measured in patients treated with L-2 and DC immunization. There was also a trend for improved CD4 + T cell responses after combination therapy.

Allogeneic immunity against neoplastic cells depends on the balance between effector T cells and regulatory T (Treg) cells. Treg cells prevent immune attack against normal and neoplastic cells by directly suppressing the activation of effector CD4 + and CD8 + T cells. Use of recombinant interleukin 2 / diphtheria toxin complex (DAB / IL2; Denileukin diftitox; ONTAK®) as a method to deplete Treg cells and destroy immune tolerance to human neoplastic tumors It was done. DAB / IL2 (12 μg / k
g; 4 times daily: 21-day cycle) was administered to 16 patients with metastatic melanoma and the effects of several T cell subsets on peripheral blood concentrations and systemic tumor tissue volume were measured.

Rasku et al. (J. Translational Medicine; 6:12 (200
8)) found that DAB / IL2 caused a temporary depletion (<21 days) of Treg cells and total CD4 + and CD8 + T cells. T cell regrowth is the renewal of melanoma antigen-specific CD8 + T cells in several patients as measured by flow cytometry using tetramer MART-1, tyrosinase and gp100 peptide / MHC complex. Consistent with the appearance. Sixteen patients received at least one cycle of DAB / IL2, and five of these patients experienced regression of melanoma metastases as measured by CT and / or PET imaging. One patient experienced an almost complete remission with several liver and lung metastasis regressions associated with the new appearance of MART-1 specific CD8 + T cells. Only one metastatic tumor remained in this patient, and after surgical resection, immunohistochemical analysis revealed MART1 + melanoma cells surrounded by CD8 + T cells. Temporary depletion of T cells in cancer patients
It may disrupt homeostatic homeostatic control and allow for expansion of effector T cells with specificity for neoplastic cells.

Recent studies have demonstrated that the lack of naturally induced tumor associated antigen (TAA) specific immunity is not just a passive process. Barnett et al. (Am. J. Reprod.
Immunol. , 54 (6) 321 (2005)) demonstrated that tumors actively suppress the induction of TAA-specific immunity by induction of TAA-specific tolerance. This immune tolerance is mediated in part by regulatory T cells (Tregs). Barnett et al. Showed evidence that deregulation in human cancers, such as ovarian cancer, using Denileukin diftitox (ONTAK®) enhances immunity.

CD4 + CD25 + Foxp3 + regulatory T (Treg) cells are involved in the lack of effective anti-tumor immunity (Litzinger et al., (2007) Blood; 11
0 (9): 3192-201). Denileukin diftitox (DAB (389) IL-
2) provides a means of targeting Treg cells. Spleen of normal C57BL / 6 mice,
Peripheral blood and bone marrow Treg cells were variously reduced after a single intraperitoneal injection of Denileukin diftitox. The decrease was evident within 24 hours and lasted about 10 days. Injection of Denileukin diftitox one day prior to immunization with other agents enhanced the antigen-specific T cell response beyond the level induced by immunization alone. Litzinger et al., In a mouse model, the different effects of Denileukin diftitox on Treg cells in various cell compartments, the advantages of combining Denileukin diftitox with other drugs to enhance antigen-specific T cell immune responses, live viral vectors The lack of inhibition of the host immune response to Dnileukin diftitox and the importance of the administration regimen of Denileukin diftitox when combined with the immunogen were demonstrated.

Tregs have been shown to be an indispensable part that regulates and possibly suppresses the immune response to growing tumor cells. Matsushita et al. (J. Im
munol. Methods; 333 (1-2): 167-79 (2008)) are three methods of Treg deficiency and / or elimination, ie, methods utilizing low dose cyclophosphamide (CY), Treg (PC61) surface. A method using a specific antibody against the found IL-2 receptor and a method using Denileukin diftitox (DD) were compared. M
Atsushita demonstrated that the use of DD resulted in> 50% Treg cell depletion without parallel killing effects on other T cell subsets, but did not enhance anti-tumor immunity against B16 melanoma. Finally, PC61 does not reduce the total number of CD8 (+) T cells,
It showed a moderate decrease in Treg that lasted longer than the other reagents.

Provided herein are methods for increasing the activity of anticancer agents (eg, RNA-transfected DCs, anti-CLTA4 antibodies, MISIIR scFvs, etc.) by administering a DT variant-IL2 fusion protein described herein. In one embodiment, a DT variant-IL2 fusion protein is administered followed by an anticancer agent. In one non-limiting example, the DT variant-IL2 fusion protein is administered at least 4 days before the anticancer agent.

In addition, anticancer agents (eg, RNA-transferred DC, anti-CLTA4 antibody, MISIIR scFv
s) and a DT variant-IL2 fusion protein described herein is provided herein for methods of treating metastatic cancer by reducing or eliminating Tregs. Examples of metastatic tumors include metastatic renal cell carcinoma, metastatic prostate cancer, metastatic ovarian cancer, and metastatic lung cancer. In one embodiment, a DT variant-IL2 fusion protein is administered followed by an anticancer agent. In one non-limiting agent, D
The T variant-IL2 fusion protein is administered at least 4 days before the anticancer agent.

In another embodiment, an anti-cancer agent (eg, RNA transfected DC, anti-CLTA4 antibody, MISII
RscFvs, etc.) and a DT variant-IL2 fusion protein as described herein is a method of treating prostate, ovarian, lung, or melanoma by reducing or eliminating Tregs. Provided in In one embodiment, D
A T variant-IL2 fusion protein is administered followed by an anticancer agent. In one non-limiting agent, the DT variant-IL2 fusion protein is administered at least 4 days before the anticancer agent.

The toxicity and therapeutic effects of such ingredients are given as LD ₅₀ (dose that causes 50% of the population to die)
And ED ₅₀ (dose that is therapeutically effective in 50% of the population) can be examined by cell culture or standard pharmaceutical procedures in laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 _/ ED _50. Compounds that exhibit large therapeutic indices are preferred. Compounds with toxic side effects may be used, but such compounds are targeted to affected affected tissue sites with the aim of minimizing possible damage to non-cancerous and / or normal cells It should be noted that the delivery system is designed to reduce the side effects.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. The dose achieves a blood plasma concentration range that includes an IC ₅₀ (ie, the concentration of the test compound that achieves half-maximal inhibition of symptoms) as measured in cell culture and as shown below in Example 4. Can be prescribed in animal models. Such information can be used to more accurately determine useful doses in humans. Plasma concentration can be measured, for example, by high performance liquid chromatography.

The embodiments of the compounds and methods of the present application are intended to be illustrative and not limiting. Modifications and changes can result in reduced immunogenicity and / or reduced HUVEC binding while remaining functionally nearly native with respect to being able to be used as a DT venom in the assembly of protein fusion toxins. One skilled in the art can make in light of the above teachings that may be involved in alterations in the DT toxin around the described modifications to the T cell epitope sequences, the described VLS sequences and the B cell epitope sequences.

Those skilled in the art will also be able to use the compounds and methods described herein for other purposes, such as the delivery of other novel molecules to selected cell populations.

This application contemplates compositions for use in immunization embodiments. Less effective in promoting VLS or other toxic effects by alterations in one or more (x) D / E (y), (x) D / E (y) T and / or flanking sequences It is contemplated that the proteinaceous composition is useful as an antigen that stimulates an immune response to a toxin. In a specific embodiment, one or more modified (x) D / E (y), (x) D / E (y
) DTs containing T and / or flanking sequences are contemplated as useful antigens. Preferably, the composition is extensively dialyzed to remove unwanted small molecular weight molecules and / or lyophilized for easier incorporation into a given vehicle. In another embodiment,
Toxins that lack one or more active site residues (ie, toxoids) can also be used for immunization.

The compounds and methods described herein deliver such substances in one or more clinical situations where it is desirable for the amount of toxin to reduce as much as possible the immunogenicity, potential of VLS or combinations thereof. Can be used under the conditions used for In this situation,
The catalytic domain or some portion thereof is substituted, inactivated and fused with the desired substance or molecule. An acid or protease sensitive cleavage site can be inserted between the remainder of the catalytic domain and the desired substance or molecule.

Substances or molecules that can be linked to the modified toxins disclosed herein include, but are not limited to, peptides or protein fragments, nucleic acids, oligonucleotides that require selective delivery,
Examples include acid insensitive proteins, glycoproteins, proteins or novel chemical entities.

Accordingly, it is to be understood that changes can be made in the particular embodiments disclosed that are within the scope described.
XI. Packages and Kits In yet another embodiment, the application relates to kits for using the compounds described above. Toxins that exhibit reduced immunogenicity, VLS promoting or toxic effects, or combinations thereof can be provided in the kit. Such kits are specific for toxins in order to generate fusion proteins that target specific receptors on the cell surface (eg, IL-2 receptors on the cancer cell surface) in ready-to-use and stored containers. Can be used in combination with specific cell binding ligands. Thus, the kit will contain in a suitable container means a composition having reduced immunogenicity, VLS promoting or toxic effects, or combinations thereof. The kit may contain the modified DT or its fusion protein in a suitable container means.

The container means of the kit generally comprises at least one vial, test tube, flask, bottle, syringe and / or other container means in which at least one polypeptide can be placed and / or preferred. Can be dispensed as appropriate. The kit can include means for placing at least one fusion protein, detectable moiety, reporter molecule, and / or any other reagent container in tight control for commercial scale. Such containers can include injection molded and / or blow molded plastic containers that house a given vial. The kit can also include printed materials for use of the kit material.

Packages and kits can further comprise buffering agents, preservatives and / or stabilizers in the pharmaceutical formulation. Each element of the kit can be enclosed in individual containers, and all the various containers can be contained in a single container. The kit of the present invention can be designed for cold storage or room temperature storage.

The formulation also contains a stabilizer (eg, bovine serum albumin (BSA)) to increase the shelf life of the kit. If the composition is lyophilized, the kit can contain another formulation of solution to reconstitute the lyophilized formulation. Acceptable reconstitution solutions are well known in the art, for example, pharmaceutically acceptable phosphate buffered saline (PBS).
Is mentioned.
Also, a package or kit provided herein may further include any of the other parts provided herein, such as one or more reporter molecules and / or 1
One or more detectable moieties / substances may be included.

The packages and kits can further contain one or more elements for assays such as ELISA assays, cytotoxicity assays, ADP-ribosyltransferase activity assays, and the like. Samples to be tested in this application include, for example, blood, plasma and tissue sections and secretions, urine, lymph and their products. Packages and kits can further include one or more elements for sampling (eg, syringes, cups, swabs, etc.).

The packages and kits can further include a label specifying, for example, product description, method of administration and / or therapeutic indication. The packages provided herein are:
Any of the compositions described herein can be included. The package can further include a label for treating the cancer.

The term “packaging material” refers to the physical structure that houses the elements of the kit. The packaging material can hold the element aseptically and can be made of materials commonly used for such purposes (eg, paper, corrugated fiber, glass, plastic, foil, ampoules, etc.). The label or package insert can include appropriate instructions. Thus, the kit can further include a label or instructions for using the kit element in any method of the invention. The kit can include the compound in a pack or dispenser along with instructions for administering the compound in any method of the invention.

The instructions can include any of the methods described herein, such as instructions for performing a therapeutic method. The instructions can further include sufficient clinical indicators or indications of possible adverse symptoms, or additional information required by a regulatory agency such as the Food and Drug Administration for use with human subjects.

Instructions are “printed”, eg, paper or cardboard affixed to or in the kit, or a label affixed to the kit or packaging material, or attached to a vial or tube containing the kit elements It may also be a labeled label. The instruction manual further includes computer readable media such as disks (floppy disks or hard disks), optical CDs such as CD- or DVD-ROM / RAM, magnetic tape, electrical storage media such as RAM and ROM. , IC chips and hybrids thereof, for example, magnetic / optical storage media.

The present invention may be better understood with reference to the following non-limiting examples provided as exemplary embodiments of the present invention. The following examples are presented for the purpose of more fully illustrating the embodiments of the invention, but should not be construed as limiting the broad scope of the invention. While certain embodiments of the present invention have been shown and described herein, it will be apparent that such embodiments are provided by way of example only. Numerous changes, changes and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein can be used in the practice of the methods described herein.

Assembly, expression and purification of DT variant and DT fusion protein Assembly of DT variant and DT fusion protein N-terminal methionine residue and amino acid residue 1-386 of native DT (SEQ ID NO: 2) (
Here, a truncated DT-type poison containing cis-residue 2-387) of the tip-cleaved toxin is designated as DT38.
Assemble as residue 1-382 of 7 or DT387. The DT-type poison is composed of an N-terminal methionine residue, amino acid residue 1-386 (SEQ ID NO: 2) of native DT (here, residue 2-387 of the tip-cutting poison), It can contain residues 484-485 of native DT and is assembled as DT389. DT387 and DT389 are residues 7-9 (
VDS), residues 29-31 (VDS) and residues 290-292 (IDS) with three (x)
Contains the D (y) motif. DT382 is a residue 1-38 of DT387 or DT389
2 is contained. Other C-terminal truncated DT constructs as described herein can be used in the assays provided herein to test the functionality of DT variants. DT389
It will be appreciated that modifications made to can also be made with a cleavage structure (eg, DT382) and can be tested for functionality. FIG. 31 shows the amino acid sequences of wild type DT382, DT382 variant, and null structure DT382 (G53E). The underlined sequence is a vector / tag sequence, the enterokinase cleavage site is highlighted in italics, and mutations from the WT sequence are shown in bold.

Site-directed mutagenesis is used to change the (x) D (y) motif of DT. Strat
The mutants are assembled using the agene Quickchange mutagenesis kit. Oligonucleotide primers are designed to change coding residues within the (x) D (y) motif involved in VLS.

SEQ ID NO: 4-147 is shown in a list of exemplary non-limiting modified DT and corresponding amino acid sequences.

Mutants are tested in connection with protein fusion toxins gene fused to human interleukin 2 (SEQ ID NO: 3) or a sequence encoding a cell binding portion thereof. DT fusion protein is expressed and purified.

Expression and purification of DT variants and DT fusion proteins Plasmid constructs encoding cleaved and / or modified DT proteins, modified DT mutants and modified DT fusion proteins are transformed into E. coli HMS174 (DE3) cells. E. coli HMS174 is a protease-deficient strain that can achieve overexpression of the recombinant protein. Induction of recombinant protein expression is obtained by adding isopropylthiogalactosidase (IPTG) to E. coli HMS174. After incubation
Bacterial cells are harvested by centrifugation, lysed, and the recombinant protein is recovered from Murphy and va
Inclusion body formulations reported by nderSpek (Methods in Molecule)
lar Biology, Bacterial Toxins: methods and
protocols, 145: 89-99 Humana press, Totowa.
, N.M. J. et al. (2000)). It may be necessary to remove endotoxin from the protein formulation to ensure that the effect on HUVEC is from VLS and not due to the presence of endotoxin. Endotoxins are <25 by passing ion exchange resins.
Removed to 0 EU / ml. Separation of degradation products from full length material occurs during ion exchange chromatography. After another final purification using ion exchange resin, endotoxin is <25
Reduced to EU / ml, the venom is tested for VLS as a function of HUVEC cell binding in vitro. For analysis of DT ₃₈₇ or DT ₃₈₉ toxicants, samples from the above methods are separated by SDS polyacrylamide gel electrophoresis (PAGE) using conventional methods described herein and known in the art. Can be performed using Coomassie Blue staining and Western blot.

Mutations resulting in stable constructs with appropriate expression that do not affect the ribosyltransferase activity of the DT ₃₈₇ or DT ₃₈₉ venom are subsequently followed by the corresponding VLS modified DT-EGF and VLS modified DT-IL-2. Protein fusion toxins (each Example 5)
Can be tested for targeted cytotoxicity.

Cell permeability assay Human vascular endothelial cells are maintained in EGM medium (obtained from Cambrex, Walkersville, Md). Subconfluent early passage cells are seeded on plastic coverslips at an equivalent T cell number. Purified wild-type D without endotoxin
T poisons and mutants were fluorescently labeled F-150 (Molecula by chemical coupling).
r Probes, Eugene, Oreg). HUVEC are incubated with an equal amount of labeled poison. The medium is then aspirated, then the cells are washed and fixed,
Prepared for analysis. Examination of cells on cover slips from different treatment groups allows an analysis of the number of cells labeled with a fluorescent toxin. The target ligand is not present on the poison body surface, so the level of HUVEC interaction is only proportional to the affinity of the poison body for HUVEC.
Comparisons were made using a fluorescence microscope by comparing the number of labeled cells from at least 10 independent regions, different coverslips or different slides. In particular in the case of mutant structures, cell labeling is not readily visible, so DAPI stain is used to localize the cells. 4'-6-diamino-2-phenylindole (DAPI) is known to form fluorescent complexes with natural double-stranded DNA. As such, DAPI is a useful tool in cytochemical studies. When DAPI binds to DNA, its fluorescence is strongly enhanced. Thus, DAPI serves as a method for labeling cell nuclei. On the other hand, F-1
Cells treated with 50DT poison are easily observed. Changes in signal intensity and background signal are also increased to facilitate quantification of mutant DT venom structures.

The effect of DT variant IL-2 fusion protein on the morphology of HUVEC monolayers is also described, for example, by Baluna et al. (Int. J. Immunopharm., 18: 355-361, 1).
996) and Soler-Rodriguez et al. (Exp. Cell Res., 206
: 227-234, 1993). DT VL
To examine whether S sequences and IL-2 damage HUVEC, monolayers are incubated with various concentrations of DT variant, DT variant-IL-2-fusion protein or control. HUVECs were isolated, cultured and examined under a microscope. In short, HUVEC
Monolayers are incubated with 10 ⁻⁶ M of each variant, fusion protein, control, or medium alone for 18 hours at 37 ° C. and then examined with a phase microscope (20 × magnification). A normal monolayer consists of highly confluent cells with an elongated shape, whereas damaged cells curl and pull away from the plate. Untreated HUVEC consists of dense, elongated cells.
Monolayers can be evaluated after 2 hours for cell spheronization after 2 hours of incubation and after 18 hours for formation of gaps in the monolayer. To assess toxic effects on HUVEC.

Another method for measuring the permeability of endothelial monolayers in vitro has already been described in detail (Fri
edman et al. , J .; Cell. Physiol. 129: 237-249.
(1986); Downie et al. , Am. J. et al. Resp. Cell. Mol.
Biol. 7 (1) 58-65 (1992)). Following incubation with the various media described, the filters containing confluent endothelial cells are washed twice with PBS. A filter with bound endothelial cells is then attached to the modified flux chamber and the chamber is placed in a culture dish. The upper well of the chamber is filled with serum free medium containing 50 mM Hepes. Fill this dish with the same medium as above. A stir bar is added to the lower well and the entire chamber is placed on an electric stirrer and incubated at 37 ° C. The chamber is incubated until the amount of medium between the upper well and the surrounding liquid in the beaker is the same. Therefore, there is no hydrostatic pressure difference between the upper well and the lower well. After this equilibration time, a small aliquot of the medium in the upper well is removed and replaced with medium containing [ ¹²⁵ I] bovine serum albumin (30,000 cpm / ml). Radiolabeled albumin is dialyzed thoroughly against 1M PBS immediately before use. Dialysis [ ^12
5 I] Simultaneous chromatograph with albumin (Friedman et al., J. Cell. Physiol.) By chromatographic monitoring of albumin and lower well media.
129: 237-249 (1986))> 95% ¹²⁵ I is demonstrated. ¹
Small aliquots (in triplicate) of medium are removed continuously from both the upper and lower wells at 10, 30, 60, 120, 180 and 240 minutes after addition of the ²⁵ I probe. [ ¹²⁵ I] activity of each aliquot is measured with a gamma counter and the average cpm / ml is determined for samples from the upper and lower wells. Make appropriate corrections for background using experimental media. [ ¹²⁵ I] albumin migration rates of BPAEC monolayers are expressed as the rate of appearance of lower well counts versus upper well / hour counts throughout the steady state clearance of 90-240 minutes (Friedma
n et al. , J .; Cell. Physiol. 129: 237-249 (198
6)). Each albumin migration rate point (“n”) represents the average rate of the duplicate filter within a group. Each group of filters contained a duplicate control filter (ie, a monolayer on a filter incubated with diluent alone). In another filter, non-radiolabeled bovine serum albumin (final concentration 1%) was added to the upper well [
¹²⁵ I] with bovine serum albumin. [ ¹²⁵ I] albumin migration rate across the monolayer is measured using the method described above. The endothelial monolayer is expected to be less damaged after exposure to the DT variant-IL-2 fusion protein compared to the unmodified DT-IL-2 fusion protein.

In yet another assay, the DT-IL-2 mutant channel-forming activity was examined using a planar lipid bilayer membrane system (vanderSpek et al.,).
J. et al. Biol. Chem. 268: 12077-12082 (1993); Silver
rman et al. , J .; Membr. Biol. 137: 17-28 (1994).
); Hu et al. , Prot. Eng. 11 (9): 811-817 (1998).
)), Compared to unmodified DT-IL-2. The membrane is 50-1 formed in a polystyrene cup.
It is formed across an opening of 00 μm. Lecithin type IIS (Sig
ma) in 1% hexane (Kagawa and Racker, Biol. Chem)
. 246: 5477-5487 (1971)) on both sides of the opening and dried. The outside of the opening is then covered with a 1.5% squalene solution prepared in light petroleum. The cup is placed in the back chamber of a block prepared by Warner Instruments (Hamden, CT). Buffer (1M KCl, 2mM
_{CaCl 2, 1mM EDTA, 50mM HEPES} , and pH 7.2), added to the top of the opening level of the cup (0.5 ml). HEPES in the front chamber of the block
The same buffer solution 1.0m except that 30mM MES (pH 5.3) is used instead of
1 is satisfied. A 50 μl aliquot of lecithin hexane solution is layered over the front chamber buffer and the hexane is evaporated. The front chamber buffer is then lowered and raised above the level of the opening to form a planar lipid bilayer. Unmodified DT-IL-2
20-730 ng of fusion protein and its DT variant-IL-2 fusion protein
Add to front chamber at a concentration of / ml. A voltage of +60 mV is applied across the membrane using potential fixation conditions. The back chamber of the block containing the cup is held in virtual ground and the voltage refers to the front chamber to which the protein is added. Current, standard method (
Jakes et al. , J .; Biol. Chem. , 265: 6984-6991 (
1989)). Use g = I / V for the conductivity of the groove, where g is the conductivity, I is the current flowing through the membrane, and V is the voltage applied across the membrane And measure. The lipid bilayer is expected to be less damaged after exposure to the DT variant-IL-2 fusion protein compared to the unmodified DT-IL-2 fusion protein.

This example illustrates a method for testing ADP-ribosyltransferase activity.
Ribosome inactivating protein toxins, such as diphtheria toxin, are peptide chain elongation factor 2 (
Catalyze the covalent modification of EF-2). Ribosylation of the modified histidine residue of EF-2 stops protein synthesis at the ribosome leading to cell death. Each ribosyltransferase assay to examine the catalytic activity of the DT ₃₈₇ mutant was 50 mM Tris-Cl (pH 8.0), 25 mM EDTA, 20 mM dithiothreitol, 0.4.
mg / ml purified EF-2 and 1.0 pM [ ³² P] -NAD ⁺ (10 mCi / ml, 1
000 Cimmol, Amersham-Pharmacia). The purified mutein is tested in a final reaction volume of 40 μl. Reactions are performed in 96 well V-bottom microtiter plates (Linbro) and incubated for 1 hour at room temperature. The protein is precipitated by adding 200 μl of 10% TCA, recovered with a glass fiber filter, and radioactivity is measured with a standard protocol. Conventional methods for measuring ADP-ribosylation use permeable cells treated with double stranded (ds) activator DNA oligonucleotides, and subsequent measurements of radiolabeled NAD ⁺ are incorporated into acid insoluble materials. . FACS
For example, Kunzmann et al. (Immunity & Aging, 3
: 8 (2006)) can also be used.

Cytotoxicity assay on crude extract of modified DT-IL-2 fusion protein cells The DT387 or DT389 construct can be used first to allow the modified venom to be chemically linked to a number of target ligands and functional Demonstrate that it produces a target toxin. DT387
Single chain fusion toxins such as those represented by linker IL-2 or DT389 linker IL-2 avoid the scale-up purification problems typically encountered in complex toxin generation. To confirm the effect of the engineered changes, a number of modified DT387 IL-2 fusion toxins or DT389 IL-2 fusion toxins are produced and tested in cytotoxicity assays.

Amino acid substitutions are made as described above, and a cytotoxicity assay is performed to see that this change does not result in an inactive carrier that cannot produce a fusion toxin.

Cytotoxicity Assay Cytotoxicity assays are performed using HUT102 / 6TG cells, a human HTLV1 transformed T cell line that expresses the high affinity interleukin-2 receptor. HUT102 /
6TG cells are maintained in RPMI 1640 (Gibco) medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 IU / ml penicillin and 50 μg / ml streptomycin. Cells are seeded in 96-well V microtiter plates at a density of 5 × 10 ⁴ / well. Fusion protein toxins are typically from 10 ⁻⁷ M to 10 ^{−12 in} wells.
Add in molar concentrations ranging up to M. The final volume of the well is 200 μl. Plates are incubated for 18 hours at 37 ° C. in a 5% CO ₂ environment. The plate was subjected to centrifugation to pellet the cells, the medium removed, 1.0 μCi / ml [ ¹⁴ C] leucine (<280 mCi / mmol, Amersham-Pharmacia) and 21 mM glutamine, 50 IU / ml penicillin and 50 μg / Replace with 200 μl of leucine-free minimal essential medium containing ml of streptomycin. The cells are shaken for 90 minutes and then the plate is subjected to centrifugation to pellet the cells. Remove supernatant and 60 μl of cells
In 0.4M KOH and then incubated at room temperature for 10 minutes. Then
140 μl of 10% TCA is added to each well and incubated for another 10 minutes at room temperature. The precipitated protein is collected on a glass fiber filter using a PHD cell harvester and the incorporated radioactivity is measured using standard methods. Results are reported as a percentage of [ ¹⁴ C] -leucine incorporation, with control (no fusion protein added to inhibit protein synthesis). Toxilight (TM), Vialight (TM) and ALAMARBLUE (TM) kits are non-radioactive commercial assays that can be used to evaluate variants. The assay is performed in a 96 well plate format and the toxin (10 ⁻⁷ M to 10 ⁻¹² M) is titrated over time using sensitive and resistant cell lines.

Pharmaceutical grade GMP purified DAB ₃₈₉ IL-2 produced from E. coli is typically 5
× results in a _{^{10 -11 M~1 × 10 -12 M IC}} 50 of. Partially purified toxins exhibit 10-100 fold lower activity in partially purified heterogeneous extracts. The pharmaceutical grade toxin is purified to homogeneity and the active fraction of the regenerated fusion toxin is used as a bioactive agent. In the above examples, we used medium throughput analysis to examine the receptor-specific cytotoxicity of partially purified modified DT-IL-2 fusion toxins, which were similarly purified DAB ₃₈₉ IL- Compared to the activity of 2. These assays are based on the modified DT ₃₈₇ linker IL-2 fusion DAB ₃₈₉ IL
Demonstrate comparable activity against -2. It should be noted that the calculation of specific cytotoxicity is based on the total amount of protein in the partial fusion toxin sample. For the assay, equimolar concentrations of fusion toxins were tested.

The relative amount of unfused toxin protein in each sample can artificially alter the IC50 of any given structure. That is, non-full length or non-fused toxin proteins in the sample used for this analysis would probably account for slight differences in IC ₅₀ .

Purified DAB ₃₈₉ IL-2 produced in E. coli is typically 5 × 10 ⁻¹¹ M-1
This produces an IC ₅₀ of × 10 ⁻¹² M.

A medium throughput cytotoxicity assay was used to analyze the crude purified DT-IL-2 fusion toxin, which was compared to the activity of similarly purified DT ₃₈₇ linker IL-2.

It should be noted that there is one (x) D / E (y) motif in IL-2 located at residues 19-21 (LDL). The contribution of IL-2 to VLS is IL-
The (x) D / E (y) motif in 2 can be examined and the modified protein tested using the cytotoxicity assay described above. For example, DT ₃₈₇ or DT ₃₈
Modified DT mutants derived from _7- linker IL-2 can be used to distinguish the effects of mutations on catalytic activity, VLS activity and effective delivery of target toxins to the cytosol of target cells . Also, a comparison between DT ₃₈₇ and modified DT mutants of DT ₃₈₇ linker IL-2 shows the effect of the modification sequence of the venom alone, the IL-2 targeting ligand present in DT ₃₈₇ linker IL-2. Will be distinguished from In another example, a modified DT mutant derived from both DT ₃₈₉ and DT ₃₈₉ linker IL-2 is used to effectively catalyze catalytic activity, VLS activity and target toxin to the cytosol of target cells. The effect of mutation on delivery can be distinguished. DT ₃₈₉ and DT ₃₈₉ linker IL
-2 between modified DT mutants shows the effect of the modified sequence of the venom alone, DT _38
A distinction will be made from the IL-2 target ligand present in the ₇ linker IL-2.

This example illustrates a method for testing the effects of the fusion proteins described herein in vivo. A vascularized human skin is transplanted into SCID mice, the mice are injected with a toxin-containing fusion protein, and the body fluid contains toxins against the human endothelium in vivo by measuring the fluid accumulation in the graft as a wet / dry weight ratio. Models have been developed to study the effects of fusion proteins (Baluna et al., J. Immunother., 22 (
1): 41-47 (1999)). Fluid accumulation in human skin is measured by measuring the weight of a punch biopsy of the skin graft before and after lyophilization. This model can be used to evaluate the effects of the modified DT fusion proteins described herein in vivo.

Fluid accumulation in the lungs of normal SCID mice is also used as a surrogate model for VLS. IL-
2 has been shown to induce fluid accumulation in mouse lungs (Orucevic)
and LaIa, J. et al. Immunother Emphasis Tumor Imm
unol. 18 (4): 210-220 (1995)). The moisture content of the lung or skin graft is calculated as a wet / dry weight ratio. In this model, even pulmonary vascular ^leak, 125 I-
Albumin is assessed by measuring accumulation in lungs injected intravenously (Smal
lshaw et al. , Nature Biotechnology, 21: 387.
-391 (2003)).

Multiplex assays, such as in vitro cytotoxicity assays, in vitro vascular toxicity, in vivo mouse models, and any other assay described herein or known in the art are described herein.
Can be used to test the function of the T variant.

In vitro cytotoxicity assay The cytotoxic activity of various modified DT fusion proteins is measured using CD22 ⁺ Daudi cells and [ ³ H] -leucine incorporation as previously described (Ghetie et
al. 1988). The concentration of fusion protein that reduces [ ³ H] -leucine uptake by 50% relative to untreated control cultures is defined as the IC ₅₀ .

Vascular toxicity As a first step to assess the ability of fusion proteins prepared with modified DT to induce vascular injury, a series of in vitro tests using HUVEC can be performed. For in vitro assays, the effect of the modified DT fusion protein on the morphology of the HUVEC monolayer is tested as previously described (Baluna et al., 1996).

In vivo assays The effect of modified DT fusion proteins can be examined in a SCID / Daudi tumor model (Ghetie et al., 1992). Briefly, female SCID mice are injected intravenously with 5 × 10 ⁶ Daudi cells on the day (IV, lateral tail vein). The fusion protein was injected on day 1, day 2, day 3 and day 4 V. Inject. Groups of 5 mice are used for each treatment and the test is repeated. Treatment groups include (1) untreated (control), (
2) receiving an unmodified DT fusion protein, or (3) receiving an unmodified DT fusion protein. The mouse is followed and sacrificed when limb paralysis occurs. Pulmonary vascular leakage in SCID mice is assessed as described (Baluna et al., 1999). Lung water content is calculated as the wet / dry weight ratio of lungs taken from mice injected with 10 μg modified DT fusion protein / mouse body weight.

This example illustrates a method for screening toxins for T cell epitopes.
The interaction between MHC, polypeptide and T cell receptor (TCR) provides a structural basis for antigen specificity of T cell recognition. The T cell proliferation assay tests the binding of the polypeptide to MHC and the recognition of the MHC / polypeptide complex by the TCR. The in vitro T cell proliferation assay of this example involves stimulation of antigen presenting cells (APCs) and peripheral blood mononuclear cells (PBMC) containing T cells. Stimulation is performed in vitro using synthetic peptide antigens. T cell proliferation stimulation, ³ H- presence of thymidine using ^{(3 H-Thy)} was measured and incorporated ^{3 H-Thy} uses scintillation counting of washed fixed cells were evaluated To do.

A buffy coat is obtained from a human blood donor pool that has been stored for less than 12 hours and a polypeptide library corresponding to the amino acid residue sequence of the toxin is synthesized by known methods or also obtained from an appropriate source. Red blood cells and white blood cells are separated from plasma and platelets by gentle centrifugation of the buffy coat. Remove the upper phase (containing plasma and platelets) and discard. Red blood cells and white blood cells are diluted 1: 1 in phosphate buffered saline (PBS), followed by 15 ml
Laminate on GE Healthcare. Centrifugation is performed according to the manufacturer's recommended conditions and PBMCs are harvested from the serum + PBS / Ficoll Park interface. PBMC are mixed with PBS (1: 1) and collected by centrifugation. The supernatant is removed and discarded, and the PBMC pellet is resuspended in 50 ml PBS. Cells are pelleted again by centrifugation and the PBS supernatant is discarded. The cells were then washed with 50 ml of AIM V
Resuspend using media, count at this point and assess viability using trypan blue dye exclusion. Cells are again collected by centrifugation and the supernatant discarded. Cells are resuspended at a density of 3 × 10 ⁷ per ml for cryopreservation. The storage medium was 90% (v / v) heat inactivated AB human serum (Invitrogen) and 10% (v / v) DMSO (Invitrogen).
rogen). Cells are transferred to a controlled freezing container and left overnight at −70 ° C. before being transferred to liquid N ₂ for long-term storage. If necessary, cells are thawed rapidly in a 37 ° C. water bath and then transferred to 10 ml of preheated AIM V medium.

PBMC are stimulated with polypeptide antigens at a density of 2 × 10 ⁵ PBMC per well in 96 well flat bottom plates. PBMC are incubated for 7 days at 37 ° C. and then shaken with ³ H-Thy. A synthetic polypeptide (15-mer) is generated that overlays 12 amino acid increments spanning the entire toxin sequence.

Each polypeptide is individually screened against PBMC isolated from 20 naïve donors. Two control polypeptides that have already been shown to be immunogenic and a potent non-recall antigen, KLH, are used in each donor assay. The control antigens used are Flu hemagglutinin; Chlamydia HSP60 peptide and keyhole limpet hemocyanin. The polypeptide is dissolved in DMSO to a final concentration of 10 mM;
These stock solutions are then diluted 1/500 in AIM V medium (final concentration 20 μM). Polypeptides are added to flat bottom 96 well plates to obtain final concentrations of 2 μM and 20 μM in 100 μl. The viability of thawed PBMC was assessed by trypan blue dye exclusion, then cells were resuspended at a density of 2 × 10 ⁶ cells / ml and 100 μl (2 × 10 ⁵ PBMC / well) was added to each well containing peptide. Move to. Triplicate well cultures are evaluated at each peptide concentration. Plates are incubated for 7 days at 37 ° C. in a humidified atmosphere of 5% CO ₂ . Cells are shaken with 1 μCi ³ H-Thy / well for 18-21 hours before harvesting on filter mats. CPM values are examined using a microplate beta top plate counter. Results are expressed as stimulation index. Stimulus index (SI) is determined by dividing the proliferation score measured for a test peptide (eg, the number of radioactivity counts per minute) by the score measured on cells not in contact with the test peptide. Be guided.

Deimmunization treatment can involve several proteins, such as plant toxins (European Patent Application Publication No. EP 1737).
961) and bacteria (International Publication No. WO2004 / 018684) have been successfully applied to generate T cell epitope-deficient variants of toxins. Deimmunization treatment techniques include, for example, EpiScreen ™ (Antitope, Ltd, Cambridge).
, UK), and is more sensitive for measuring T cell epitopes.

T cell epitopes are more complex proteins (Composi) than point mutations.
can be removed by substitution with sequence segments from other proteins using te Protein) ™ technology (Antitope, Ltd), resulting in T
Cellular epitopes provide a solution that is more variable in removing while retaining protein function.

The method described herein consists in generating DT variants with reduced VLS and reduced immunogenicity using the following seven steps.

Stage 1-DT T-cell epitope mapping and selected ligands such as human IL
-2;
Step 2-Assay for VLS and DT activity;
Step 3-Gene synthesis, expression and purification of total DT in E. coli in a format suitable for screening a large number of variants (approximately 100-250 variants);
Stage 4-Generation and testing of DT variants for reduced VLS (HUVEC binding assay)-Test variants twice (single locus / multilocus);
Stage 5—Generation and testing of DT variants after removal of T cell epitopes—Variants are tested multiple times (single epitope, multiple epitope, optimization), the second test is VL
Including combinations with S variants (step 4);
Step 6—Lead DT variant from step 5 and protein ligand, eg human IL
-Generation of lead DT-fusion variants by fusion with -2 and protein purification / testing (this step is optional); and step 7-EpiScreen ™ (control of wild type truncated DT (ΔR))
Immunogenicity test of lead DT1-389 (“truncated DT (ΔR)”) variant.

Testing of DT variants containing mutations in the VLS motif in the HUVEC binding assay and testing of all DT variants for efficacy in the IVTT assay, eg, wild type receptor binding (R) domain (DT (ΔR)) Without using a truncated DT variant. Test the lead for efficacy in cytotoxicity assays, with the full-length DT version,
DT (DR) is used with both fusion proteins fused to IL-2. For EpiScreen ™ confirmation of one or more lead DT variants (step 7), optimized DT variants with modified C and I domains are tested by expression of truncated DT (ΔR).

EpiScreen ™ T cell epitope mapping of DT
[Donor selection for EpiScreen]
Addenbrook's Ho Area Studies Ethics Committee
UK National Blood Service in accordance with approvals granted by the Spital Local Research Ethics Committee
od Transfusion Service) (Addenbrook Hospital, Cam
Peripheral blood mononuclear cells (PBMC) are isolated from buffy coats (from blood collected within 24 hours) from healthy community donors obtained from bridge, UK). PBMC from the buffy coat to Ly
Isolated by mphoprep (Axis-shield, Dundee, Scotland) density gradient centrifugation and CD8 + T cells are depleted using CD8 + RosetteSep ™ (StemCell Technologies, Inc). Donors were treated with a biotest HLA SSP-PCR-based histocompatibility kit (Biot
est, Landsteinerstraβe, Denmark)
It is identified by identifying the R haplotype. In addition, T against the control antigen, keyhole limpet hemocyanin (KLH) (Pierce, Rockford, USA)
The cellular response was also examined. A group of 54 donors was designated as a social group (world population).
Select to best represent the number and frequency of HLA-DR allotypes expressed in n) (Table 1 and FIG. 1). For allotypes expressed in social populations, an analysis of allotypes expressed in said populations achieves a range of> 80% and all major HL
It has been shown that A-DR alleles (individual allotypes with a frequency> 5% expressed in the social population) are well presented. Table 1 shows a comparison of the donor haplotype and the response to KLH obtained during treatment and isolation of donor PBMC (Study 1) and the donor response obtained after re-testing during this study (ANG01). A summary of donor haplotypes is shown in Table 2, and a comparison between the frequency of donor allotypes used in the study and the frequency present in the social population is shown in FIG.

Table 2. Donor details and haplotypes. Donor response (SI) to KLH is shown for two independent studies. Test 1 was performed on freshly isolated PBMC and ANG0
1 is a retest in this test. Same results for both tests (ie positive or negative)
Responses that did not produce are highlighted in gray. Three donors with very low basal cpm (<150 cpm) were excluded from the analysis.

[EpiScreen analysis: proliferation assay]
EpiScreen ™ was used to test overlapping peptides derived from the sequence of DT1-389 containing the C and I domains of DT with 10 amino acids of human IL-2 at the C-terminus. 10 amino acids of C-terminal human IL-2 (DT1-389 / I
Along with L-2 2-10), an overlapping peptide spanning residues 1-389 of DT-I was designed. A series of 128 × 15 mer peptides overlapping up to 12 amino acids, 1 × l
Combined with tetramer and 1 × 11 mer, EpiScr from a population of 51 healthy donors
Peripheral blood mononuclear cells (PB) derived using een ™ T cell epitope mapping
MC) was used to stimulate. Individual peptides were tested in replicate cultures and responses were evaluated using a T cell proliferation assay to identify the exact location of the epitope. PBMCs from each donor were thawed, counted and evaluated for viability. Cells were incubated at room temperature with AIM V medium (
Invitrogen, Carlsbad, Calif.) After which the cell density was adjusted to 2.5 × 10 ⁶ PBMC / ml (proliferating cell stock). Peptides were treated with DMSO (Sigma-Aldrich, St Lou) to a final concentration of 10 mM.
is, MO, USA). Peptide culture stocks were then prepared by diluting in AIM V medium to a final concentration of 5 μM. For each peptide and each donor, a 6-fold repeat culture was established by adding 100 μl of peptide culture stock to 100 μl of expanded cell stock in a flat bottom 96 well plate. Both positive and negative control cultures were also established in 6 replicates. All 9 × 96 well plates were used for each donor, each plate sufficient to test 15 peptides with one negative control (carrier alone) in 6 replicates. A positive control KLH was added to the final plate.

Cultures were incubated for a total of 6 days, after which 0.5 μCi of ³ [H] − was added to each well.
Thymidine (Perkin Elmer®, Waltham, Massachu
setts, USA). The culture was incubated for an additional 18 hours, after which it was harvested on a filter mat using a TomTec Mach III cell harvester. The count per minute (cpm) for each well was calculated using the MicroPlate B
eta Counter (Perkin Elmer®, Waltham, M.)
in assachusetts, USA), in parallel, in low background count mode, Meltilex ™ (Perkin Elmer®, Walta)
m, Massachusetts, USA) Measured by scintillation counting.

For proliferation assays, stimulation index corresponding to or greater than 2 (SI ≧ 2) (
An experimental threshold of SI) has already been established, whereby a sample that induces a proliferative response that exceeds this threshold is considered positive (if included, the boundary SI ≧ 1.9).
0 is emphasized). Extensive assay developments and past studies have revealed that this is a minimum signal-to-noise threshold that allows maximum sensitivity without detecting multiple false positive responses. A positive response is defined by the following statistical and experimental thresholds.

1. Significance of response by comparing test well cpm and media control well using Student's t-test of two unpaired samples (p <0.05).

Stimulation index greater than 2.2 (SI ≧ 2), where SI = average of test wells (cpm
) / Mean (cpm) of medium control wells.

Intra-assay variability was also expressed as the coefficient of variation and standard deviation (SD) of raw data from duplicate cultures.
) Was calculated.

Proliferation assays were set up in 6-fold replicate cultures (“Unadjusted Data”). To ensure that the intra-assay variability is low, the data is maximal and minimum cpm values ("adjusted data"
), And the donor response SI was compared using both datasets. Details of donor SI from both the adjusted and non-adjusted data sets are shown in FIGS.
3 shows. T cell epitopes were identified by calculating the average frequency of responses to all peptides in the test + 2 × SD (background response rate). Any peptide that has induced proliferation beyond this threshold is considered to contain a T cell epitope.

[Computer iTope ™ analysis of peptides]
The sequence of the peptide that was positive in the proliferation assay was determined by Antitope's predicted iTop (
(Trademark) software was used for analysis. This software predicts a favorable interaction between the amino acid side chain of the peptide and a specific binding pocket within the MHC class II binding groove. The arrangement of critical binding residues was examined by generating a 10-mer peptide superimposed by a single amino acid spanning over a long peptide sequence. Each decamer is
Tested against the MHC class II allotype (32 total) Antitope database and scored based on its fit and interaction with MHC class II molecules. Peptides that produced high binding scores for multiple alleles were considered to contain the core nonamer. Such tests include (a) DT1 -389 / IL-2 2-10 DT1 -389 / I
L-2 2-10 complete T cell epitope map, (b) assessment of potency of individual T cell epitopes and prioritization for removal from DT, and (c) T cell epitope association with MHC class II allotypes A comprehensive T cell epitope analysis of DT1 -389 / IL-2 2-10 including gender evaluation was provided.

[Identification of T cell epitopes]
All 130 peptides identified using the EpiScreen ™ analysis described above were extracted from 51 healthy donors (54 donors were initially selected, but donors 13, 20 and 38 had low basal cpm I.e. cpm lower than the cut-off value of 150 cpm
Have been successfully synthesized to test against. A positive response is defined by the donor that produced a significant (p <0.05) response with SI ≧ 2 for a given peptide. Borderline response (significant with SI ≧ 1.90 (p <0.
05) Response) was also included. The results of the unadjusted data analysis and the preparation data analysis were compared to confirm low intra-assay variability and that the positive response was not the result of pseudogrowth in individual wells. The results of each analysis showed little difference between the methods. The T cell epitope map was therefore compiled using adjusted data analysis. Donor stimulation indices from both unadjusted and preparative analyzes are shown in FIGS. 12 and 13, respectively. The T cell epitope is the mean frequency of responses to all peptides in the test plus twice the standard deviation (
(Referred to as “background response rate”). This is 5.6
% And was equivalent in inducing a positive response in 3 or more donors. Peptides that induce a proliferative response above this threshold were considered to contain T cell epitopes.

Table 3 provides a summary of individual donor responses for each of the peptides. A graph representing the frequency of response to the peptide can be found in FIG. Nine peptides induced a proliferative response above the background response rate (peptides 2, 31, 35, 39, 40,
41, 42, 49 and 100) and therefore considered to contain T cell epitopes. From these nine peptides, a total of seven T cell epitopes are identified within the DT-1 sequence and are discussed in more detail below.

Table 3. Summary of donor response to individual peptides. Positive response (SI ≧ 2 and p <0.0
5) is indicated by the donor number, and the individual SI is indicated in parentheses next to the corresponding donor. Borderline responses (SI ≧ 1.9 and p <0.05) are indicated by (*). The background response rate was 5.6%, equivalent to 3 donors.

[T cell epitopes identified by EpiScreen]
(A) Epitope 1-peptide 2
Peptide 2 (DDVVDSSKSFVMENF; SEQ ID NO: 159) is shown in Table 2 and FIG.
Contains a T cell epitope as shown in FIG. A total of 3 donors (30, 36 and 47) responded to peptide 2, but the response of donor 30 was borderline (1.92 SI)
). As discussed throughout the above specification, elimination of T cell epitopes can be achieved by modification of the amino acids of the identified epitopes. Modification of amino acid residues associated with binding of the epitope amino acids, eg, anchor pockets (p1 and p9) of the MHC class II binding gap, can affect the successful presentation of the epitope on MHC class II molecules. And / or prevent (ie, eliminate epitopes). Similarly, amino acid residues involved in binding the internal pocket of the MHC class II binding gap,
And / or modification of residues outside the core 9-mer epitope that affects or interacts with MHC class II molecules can eliminate the epitope. Various combinations of amino acid residue modifications for the elimination of T cell epitopes can be prepared and tested by the methods described herein.

Peptide 2 was analyzed for possible 9-mer MHC class II binding registers using Antitope's computer iTope ™ MHC class II prediction software. The software sets the most preferred binding register for the epitope based on the number of alleles that have the potential to bind (from a total of 32), and sets the average allele binding score (in this case the positive threshold is set to 0.5). To predict together. The results of this analysis show that the core 9-mer has VDSSKS with valine (V8) as a possible p1 anchor residue.
It was shown to be FVM (SEQ ID NO: 161) (FIG. 3). In this conformation, the analysis predicted 25 bonds out of 32 alleles.
A VDSSKSFVM core nonamer (SEQ ID NO: 161) is also present in both peptide 2 and overlapping peptides 1 and 3. Interestingly, donor 36 is peptide 2
And 3 (SI of 2.13 and 2.02 respectively) and statistically significant (p <0.05) with high response to peptide 1 (SI = 1.88) And
Indicates that VDSSKSFVM (SEQ ID NO: 161) is a core 9-mer binding register. Similarly, donor 30 responds to peptide 1 with an SI of 1.64, which is between 1.9 and 2.0.
Was clearly lower than the cut-off of, but was statistically significant (p <0.05) and higher than the overall background SI. Donor 47 did not initiate a positive response to peptides 1 and 3. This is probably due to the arrangement of the core nonamer within the peptide. The interaction of residues outside the 9-mer binding register is a peptide: MHC class I
It is well documented to support the stability of the I complex (Engelhard et al.
al. , 1994). Thus, peptide 2 appears to contain the core 9-mer in the optimal configuration for MHC class II binding. Review of the crystal structure of DT-1
t al. , 2000) revealed that the p1 valine residue (V8) is in a partially exposed position. Thus, polar substitution residues can be selected such that the polar portion is surface exposed and the hydrophobic region is obscured. Such modifications to T cell epitopes can reduce the immunogenicity of the toxin.

(B) Epitope 2-peptide 31
Table 3 and FIG. 2 show that four donors (donors 12, 23, 30 and 36) show the presence of a T cell epitope in this region, Peptide 31 (KVLALKVDNAETIKK; SEQ ID NO:
162). In addition, by analyzing overlapping peptides, donor 2
3 produces a borderline response to peptide 32 and donor 36 also responds to peptide 32, which response is subthreshold (SI = 1.81) but statistically significant (p <0.05) It was revealed that. Using iTope ™ computer analysis, the core nonamer of this peptide is VD with valine (V97) as the major p1 anchor residue.
It was predicted to be NAETIK (SEQ ID NO: 164) (FIG. 4). This is the response of donor 23 to peptide 32 and peptide 32 that contained the same core nonamer of donor 36.
Supported by a subthreshold response to (Figure 5). In this conformation, the analysis is
28 out of 32 MHC class II alleles were shown. Structural and homology modeling revealed that the V97 configuration is well exposed on the surface of the molecule and is not associated with the active site.

(C) Epitope 3-peptide 35
Peptide 35 (SEQ ID NO: 166) contains a T cell epitope as shown by the three donors (donors 1, 2 and 35) that produced a positive proliferative response after stimulation as described above in step 1 (donors 1, 2 and 35). FIG. 2 and Table 3). Analysis by iTope ™ revealed that the most preferred binding register for this peptide is LGLSLTEPL (SEQ ID NO: 167) with leucine (L107) as the major anchor residue, p1 (FIG. 5). . This 9-mer had the potential to bind 13 of the 32 MHC class II alleles. Donor 35 also contained peptide 3 which contained the same core nonamer
4 had a proliferative response (SI = 1.83). This was lower than the cut-off 1.9-2.0 for positive responses but was statistically significant (p <0.05) and the sequence LGLSLTEPL (SEQ ID NO: 167) is an epitope within this region Indicates that

Crystal structure analysis and homology modeling of the p1 anchor for this epitope show that it is largely obscured and a small amount is surface exposed. For epitope 1, a polar residue could be substituted to remove binding to MHC class II. The p9 anchor of this peptide is also obscured, so the changes are thought to be in other pocket positions containing p6 and p7 that are well exposed to the surface of the toxin.

(D) Epitope 4-peptide 39
Three donors (donors 5, 15 and 50) were identified as peptide 39 (LMEQVGTEEFI
A positive proliferative response was generated after stimulation with KRFG; SEQ ID NO: 168), but donor 50
Response was a borderline (SI of 1.94) (Figure 2 and Table 3). The T cell epitope within this peptide is determined by MEQVGTEEF (SEQ ID NO: 1 after iTope ™ analysis).
69) was predicted to contain as a core 9-mer MHC class II binding register (FIG. 6).
). In this conformation, the methionine at position 116 forms a p1 anchor residue, and this epitope is predicted to bind 19 of the 32 MHC class II alleles. Positions 1 and 9 of this MHC class II binding register are obscured in the core of the toxin's catalytic domain and fill each other. These residues are therefore considered important for the overall stability of the protein. As with epitope 3, residues that interact with other pockets of the core nonamer, such as p6 and p7 (which are well exposed) are considered for substitution.

(E) Epitope 5-peptide 40, 41 and 42
Potential T cell epitopes are peptides 40, 41 and 42 (SEQ ID NO: 170-172).
) Seems to be in. For all detected epitopes, the epitope in this region is
It is most immunogenic as shown by its ability to induce a response in a total of 8 different donors, representing 15.7% of the test population (Table 3 and FIG. 2). Six donors were peptide 40 (donors 15, 36, 46, 47, 49 and 50), peptide 41 (donor 3
5, 36, 40, 46, 49, 50) and 42 (donors 35, 36, 40, 47, 49 and 50), and responses to all three overlapping peptides are donors 49 and 5
Observed for 0, while donors 30, 40, 46 and 47 responded to two overlapping peptides. By iTope ™ computer analysis, the binding motif for this peptide is FI with phenylalanine (F124) as the P1 anchor residue.
It was predicted to be KRFGDGA (SEQ ID NO: 173) (FIG. 7). This 9-mer was present in all three overlapping peptides and was predicted to bind 23 out of 32 MHC class II alleles. Phenylalanine 124 is substantially obscured within the core of the catalytic domain and packs against M115 and V118 and is therefore considered potentially structurally important. However, anchor positions 4, 6, 7, and 9 are exposed to varying degrees and can be targeted as targets to remove epitopes.

(F) Epitope 6-peptide 49
Peptide 49 (SSSVEYINNWEQAKA; SEQ ID NO: 174) also contains a T cell epitope. FIG. 2 and Table 3 show three donors (donors 30, 39 and 49)
Shows that a positive proliferative response occurred after stimulation with peptide 49. iTope ™
Using computer analysis, the most preferred binding register for this peptide is 32
VEYINNW with the potential to bind 22 of the MHC class II alleles
It was predicted to be EQ (SEQ ID NO: 175, FIG. 8). This combined register is the valine (
V148) as the p1 anchor residue. Peptides 48 and 50 overlapping peptide 49 also contained the same core nonamer, but donors 30, 39 and 49 were
Responsive to overlapping peptides, residues outside the core nonamer also demonstrate that they are important for peptide binding to MHC class II molecules. Valine 148 is partially surface exposed, so the polar substitution residue can be selected such that the hydrophilic portion is surface exposed and the hydrophobic region is masked.

(G) Epitope 7-peptide 100
Epitope 7 is peptide 100 (LEKTTAALISPLGIG, SEQ ID NO: 17
7) Found within. Four donors (donors 1, 15, 30 and 35) are peptides 100
And the response of donor 15 was borderline. iTope ™ analysis predicted that the core 9-mer binding register of this peptide was LEKTTAALS (SEQ ID NO: 178) with leucine (L298) as the p1 anchor residue (FIG. 9).
This 9-mer was predicted to bind 24 out of 32 possible MHC class II alleles. This 9-mer is also present in peptide 99, and donor 30 initiates a statistically significant (p <0.05) subthreshold response (SI = I.84) to it, thus Supports LEKTTAALS (SEQ ID NO: 178) as a binding motif. Leucine 298 is well exposed on the surface of the translocation domain and is not associated with the activity of this domain. Therefore, it has no obvious problem with substitution. Thus, residues can be easily substituted for the purpose of modifying / eliminating T cell epitopes.

[T cell epitope and allotype response]
Table 4 summarizes the details of the seven T cell epitopes found within the DT-1 sequence, FIG.
Represents the location of the epitope within the protein sequence. No epitope was detected in the junction between DT-1 and IL-2.

A T cell epitope (Epitope 5) located in peptides 40, 41 and 42 induced a response in 15.7% of the test population. Epitopes located in peptides 31 and 100 (Epitope 2 and 7, respectively) induced a response in 7.84% of the test population (Table 4).
). Epitope within peptide 2 (epitope 1), epitope of peptide 35 (epitope 3), epitope within peptide 39 (epitope 4) and epitope within peptide 49 (epitope 6) responded in 5.88% of the test population. Induced. Analysis of the magnitude of the positive response (average SI) for each of the epitopes indicated that all seemed to be fairly equal in their effectiveness with an average SI of 2-2.5. However, Epitope 3 had a larger average SI of 3.13.

Table 4: Summary of magnitude and frequency of positive responses to peptides containing T cell epitopes

The association between HLA-DR allotype expression and donors responding to individual peptides was also analyzed. Epitopes 2, 5 and 7 (peptides 31, 40-42 and 100) were evaluated. Table 4
And FIG. 12 represents the frequency of allotypes expressed by donors responding to each of the peptides compared to the frequency of allotypes expressed in the ANG01 test population. An association between MHC class II allotypes and response to specific epitopes is observed when the frequency of allotypes in the responding population is more than doubled (ie, frequency in the test population × 2). It was.

EpiScre against MHC class II allotypes expressed by individual donors
Comparison between the positive donor responses observed in the en (TM) assay indicated that donors responding to epitopes 2, 5 and 7 are associated with specific allotypes. T for Epitome 5
The cellular response was associated with the expression of HLA-DRB1 ^* 11, HLA-DRB1 ^* 15 and HLA-DR5. The frequency of donors responding to this epitope and expressing these allotypes was 12%, 15% and 15%, respectively. All of these exceeded twice the frequency of these allotypes within the ANG01 study population (respectively 5%, 7% and 7%). In addition, iTope ™ analysis of the peptide revealed that HLA-DR5 binds the predicted core nonamer. T for both epitopes 2 and 7
The cellular response appears to be related to HLA-DRB1 ^* 15 and HLA-DRB5.
Responds to epitope 2 or 7 and HLA-DRB1 ^* 15 and HLA-DRB
The frequency of donors expressing 5 is 20% and 21% for each allele,
This is a 7% frequency 2 observed for each allele in the ANG01 test group.
It was over twice. ITope ™ analysis of these peptides revealed that the predicted binding registers for each peptide have the potential to bind HLA-DR5. In addition to identifying T cell epitopes, these analyzes also show an association between T cell responses to specific peptides and expression of MHC class II alleles.

Table 5. Frequency of responding donor allotypes compared to the frequency of allotypes expressed in the study population (
Expressed as%). The relationship between MHC class II allotype and response to epitope is highlighted in gray.

[VLS assay]
A variety of assays can be used to assess the VLS activity of the DT variants described herein.

In one assay, DT-IL2 or variant was conjugated to fluorescein. HUV as a surrogate for VLS activity using fluorescence microscopy / manual counting of labeled cells
Direct binding to EC cells can be assessed.

Correlations with in vivo tests, morphological changes, loss of cell monolayer permeability or cell membrane integrity, and CD31 expression represent a variety of means by which the variants described herein can be detected, and VL
It can also be used to assess S activity.

(A) Detection Using Antibody DT specific antibody can be used to detect DT bound to the endothelial cell surface by the VLS motif. Common epitopes were directly labeled to detect binding differences between variants. An ELISA was used to confirm the specificity / affinity of available anti-DT antibodies.
In addition to the methods described herein, His-tagged Abs provide another means by which DT variants can be selected.

FIG. 15 illustrates binding of DT variants (DT-Glu52; CRM mutant) to HUVEC cells and detection with antibodies using FACS analysis.

(B) Direct binding DT variants described herein can be evaluated using direct complexes of DT and toxin mutants to a fluorescent dye (Alexa488). Alexa dyes are highly stable and bright. Small protein labeling kit (Microscale Protein Lab)
elling Kit (Molecular Probes / Invitrogen: A
30006) allows for microscale labeling of proteins (20-100 μg). The degree of labeling (DOL) can be optimized and the dye: protein (D: P) ratio is measured with a spectrophotometer and is reproducible. Measure fluorescence using FACS.

The assay was performed using ONTAK-A that binds to HUVEC cells, as illustrated in FIG.
Good detection of 1488 and control DT-Glu52-A1488 was achieved. The assay achieved better detection of ONTAK-A1488 and control DT (ΔR) -A1488 binding to HUVEC cells compared to BSA-A1488 binding, as illustrated in FIG.

HUVEC cells were tested for IL-2R expression by FACS. ONTAK-A1488 binding to HUVEC cells was confirmed to be independent of IL-R as shown in FIG.

(C) Cell membrane integrity Cell membrane integrity can be assessed to determine loss of cell membrane integrity after exposure to toxins. VLS
Peptides containing the motif are described in Baluna et al. (PNAS USA, 96: 3957 (
1999)) was used to bind directly to the fluorescent dye. Alternatively, other assays described herein or known in the art can be utilized.

Cell membrane integrity assays were performed using propidium iodide (PI) and the effects on the toxin were evaluated by FACS to measure PI uptake as a surrogate for loss of cell membrane integrity after brief incubation with the toxin. (FIG. 18).

The HUVEC cell DT binding assay is tested for the possibility of inducing VLS using a cleaved DT (ΔR) containing only the C and I domains. DT molecule is HUVE
Label for analysis of binding to C. This process takes advantage of the expression of DT (ΔR) from step 3 and thus initiates the generation of the HUVEC assay after DT expression.

[DT activity / cytotoxicity assay]
An assay suitable for measuring DT activity has already been established. A cytotoxicity assay is used to confirm the DT-IL2 T cell epitope and the toxic activity of the VLS variant lead selected in the IVTT assay.

(A) In vitro transcription / translation (IVTT) assay Using an in vitro transcription / translation (IVTT) assay to measure DT activity, DT
Direct transcription / translation of the gene was tested using a rabbit reticulocyte lysate system. Typically this involves ligated transcription / translation of the luciferase gene by a chemiluminescent assay that measures the IVTT of the target plasmid (T7-luciferase), and the active toxin suppresses and reduces the luciferase signal. All variants were compared to WT DT in the same 96-well assay plate utilizing a PCR product that allows medium throughput screening (MTS) by titration curve. The surrogate IC ₅₀ can be measured from the inhibition curve.
This will result in inhibition of luciferase production for the active DT variant, since DT binds and results in covalent modification of elongation factor-2 (EF-2). Linked transcription / translation assays can be used for analysis of ribosome-inhibiting proteins to obtain information regarding the activity of the DT C domain. For example, U.S. Pat. Nos. 5,976,806 and 5,692.
Various useful for performing such ligated transcription / translation assay reactions as described in US Pat. No. 5,983, each of which is incorporated herein by reference in its entirety. These methods are known to those skilled in the art.

Using the IVTT assay described herein, the WT DR DT reaches 90% T
A dose-dependent inhibition of transcription / translation of the 7-luc plasmid was demonstrated, whereas the null was shown to be constant at about 20% inhibition (FIG. 14).

(B) Luminescent cytotoxicity assay Toxilight ™, Vialight ™ and ALAMARBLUE (
The TM kit is a non-radioactive commercial assay that can be used to measure cytotoxicity. The assay is performed in a 96-well plate format and titrates toxin (10 ⁻⁷ to 10 ⁻¹² M) over time using a sensitive and resistant T cell line. ONTA against human T cell lines
The cytotoxicity of K and IL-2 was evaluated for 48 hours using the Toxilight kit. 1
The fluorescence count per second (LCPS) reflects the degree of adenylate kinase release (FIG. 19).

(C) Ribosyltransferase assay In addition to the ligated transcription / translation assay, a ribosyltransferase assay (eg, as described in Example 3) was established in a 96-well format. This assay is based on DT in E. coli.
A sample of DT from gene expression (step 3) is used and tested simultaneously with the ligated transcription / translation assay described above. The conventional method for measuring ADP ribosylation is the double-stranded (ds) activator D
Permeabilized cells treated with NA oligonucleotides are used and subsequent measurement of radiolabeled NAD + is incorporated into the acid insoluble material.

New FACS-based methods such as Kunzmann et al. (2006 Immuni
ty & Aging) can also be used.

For the measurement of DT variant cytotoxicity, a cytotoxicity assay (as described in Example 4) has been developed and HUT102-6TG cells in a 96 well format for analysis of full length DT variants. (HUT102-6TG uses lead DT
-The cell line used for the final analysis of IL-2 protein (step 6)). Since the cytotoxicity assay requires expression of full-length DT, this assay is used after stage 3 DT expression.

Gene synthesis, expression and purification The DT and human IL-2 genes can be synthesized at the beginning of step 3 using conventional techniques known in the art, using codons optimized for expression in E. coli. Synthesized using. Full length D to be used in analysis of cytotoxicity and HUVEC binding assays (see step 2)
In order to generate T and DT (ΔR) (containing only C and I domains), a vector system containing a secretory leader sequence for export of DT into the periplasmic space of E. coli is used. Plasmids and E. coli strains are optimized to produce a soluble product. The His-labeled DT product is purified on a Ni-IDA column, and the spin column allows parallel purification of reads. As a method for purifying DT, for example, an affinity label fused with DT (for example, H
is6 labeling). The method developed in Step 3 is reliable for a number of DT variants with similar qualities so that the activity of the following variants can be accurately compared for the purpose of identifying lead candidates in Steps 4 and 5 Provide manufacturing.

Design and assembly of VLS variants of DT Variants of DT for reduced likelihood of inducing VLS were transformed with two mutations, ie each of the three (x) D (y) motifs of the first DT. Separate mutations, and 2
A combination of the second lead mutation and any additional mutations. Each D
T variants are tested in the HUVEC binding assay (stage 2) and the optimal mutation selected after the second mutation is combined with the T cell epitope mutation in the middle stage of stage 5 of the project. Generation of DT variants for these assays is due to the expression of truncated DT (ΔR) in E. coli (step 3).

Design, assembly and testing of T cell epitope variants of DT Variants of DT to eliminate T cell epitopes to reduce immunogenicity
Generated by two substitutions in the C and I domains. The first variant contains distinct substitutions at a single epitope locus, which are then combined in the second variant to produce a combination of two, three or more variant loci (
Depends on the number and priority of T cell epitopes). The second variant includes the combination with the VLS variant from stage 4. If the additional step of combining the VLS mutant with a T cell epitope substitution requires further optimization of the lead DT variant, any third round DT variant is included.

Substitution of DT at the T cell epitope locus replaces (mainly) amino acids in the core MHC-binding 9-mer derived from T cell epitopes with amino acids that occur in other proteins, particularly homologous loci of DT-related proteins, and so on. It is generated by using sequence segments from other proteins having the following tertiary structure: Analysis by peptide MHC class II binding prediction computer software iTope ™ (Antitope, Ltd.) is used as a guide for selected substitutions for the elimination of T cell epitopes. DT
Structural analysis of the crystal structure is also used as a guide for mutations that are most unlikely to reduce the activity of DT or reduce the stability of the DT structure. In some cases, such structural analysis may suggest compensatory substitutions outside the T cell epitope locus that can be adapted to certain substitutions within the T cell epitope without loss of activity or stability.

The DT T cell epitope variant primarily uses the rabbit reticulocyte assay (step 2) for analysis of substitutions within the C domain and the cytotoxicity assay (step 2) for analysis of substitutions within the C and I domains. And test. Expression of DT variants for these assays uses transcription / translation and expression of full length DT in E. coli, respectively (step 3).
For reads from the second (and optional third) substitution, ribosyltransferase and HUVEC binding assays are also used to identify lead candidates.

Twenty-six epitope variants have been designed and assembled (Figure 21). Ten out of 26 (10/26) variants have been tested in the IVTT assay as described herein. T cell epitopes are prioritized based on relative intensity (FIG. 22). FIG. 23 represents representative results for 7 epitope mutants,
Demonstrate that mutants have been obtained for each epitope that retains wild-type activity.
DT epitope variants inhibit protein synthesis The DT382 structure is used and amino acid residues 1-38 of DT SEQ ID NO: 2 or 149
2 and IL2. A restriction enzyme site was engineered at amino acid residue 382 for cloning in either the R domain or the IL2 portion. Modifications are incorporated as described below.

Variants of the DT382 gene were generated in which T cell epitopes 1, 3 and 5 were modified with a putative MHC class II binding p1 anchor residue. Residues V7, L107, and F124 of the wild-type DT sequence were replaced with amino acids (due to disruption of MHC class II binding) that were predicted to remove T cell epitopes while retaining activity. Identified as active in epitope variants). The activity of single and combined variants was measured in an in vitro transcription / translation assay. Purified PCR products of each variant were used for rabbit reticulocyte lysate, TnT buffer, T7 RNA polymerase, amino acid mixture-Met, amino acid mixture-Leu and RNasin (# N2511 Pr
Omega, Madison WI) TnT-linked transcription / translation reaction mixture (#L
4610 Promega, Madison WI, following manufacturer's instructions) using a DNA range of 1 ng to 64 ng per reaction in a total volume of 10.5 μl. The reaction was incubated at 30 ° C. for 30 minutes to allow DT between the various variants.
Allowed possible differences in the rate of gene translation. 250 ng of T7-luciferase control plasmid was then added and the reaction was incubated at 30 ° C. for an additional 45 minutes. Luciferase expression can be determined using manufacturer's instructions (# E2510 Promega, Madi
The reaction was measured by fluorescence after incubation with SteadyGlo luciferase assay reagent according to (son WI). Luminescence reading, BMG FLUO
star OPTIMA fluorescent plate reader (BMG Labtech, Durham)
, NC).

Twenty-eight VLS mutants have been designed and assembled. 18 of 28 (
18/28) VLS mutants have been tested in IVTT assays. Known VLS variants have been shown to have activity comparable to or greater than wild type (WT) and numerous alternatives demonstrating activity comparable to or greater than wild type (WT) VLS variants have been identified (Figure 20).

The% inhibition of protein synthesis was plotted against the DNA concentration in the reaction and the resulting curve was used to calculate the IC ₅₀ for each variant. In order to allow IC ₅₀ to vary within the assay, a value of = 1 represents an activity comparable to the wild type, a value of> 1 represents an increase in DT activity and a value of <1 represents a decrease in DT activity Was normalized by dividing the IC ₅₀ of wild-type DT (included in every assay plate) by the IC ₅₀ of the DT variant.

Table 6: IC ₅₀ data for modified DT variants compared to wild type and null DT variants. Data for a single variant is shown as a line graph in FIG. 27 and a quadruple variant is shown as a bar graft in FIG.

Quadruple variant V7D V97A L107N F124H, V7D V97T L1
07N F124H, V7N V97A L107N F124H and V7N V97T
L107N F124H showed an equivalent IC ₅₀ activity compared to WT.

Quadruple variant V7D V97D L107N F124H, V7N V97D L1
07N F124H, V7T V97A L107N F124H, V7T V97D
L107N F124H and V7T V97T L107N F124H showed improved IC ₅₀ activity compared to WT.

Table 7: Relative IVTT scores of VLS and / or T cell epitope variants compared to WT. The relative IVTT score is determined by dividing the IC _{50 of the} wild type DT by the IC ₅₀ of the mutant DT.

FIG. 24 represents the relative activity of the DT382 epitope variant compared to wild type DT382 in inhibiting protein synthesis. Data are for the following T cell epitope variants of DT:
V7N L107N F124H, V7N L107A F124H, V7D L107
N F124H, V7D L107A F124H, V7T L107N F124H and V7T L107A F124H and V7T V29T I290T all show similar activity to wild type DT382 in inhibiting protein synthesis. In contrast, G53E
Substitution results in decreased activity. As described herein, references to G52 modifications are
Refers to the amino acid residue numbering of the DT molecule of SEQ ID NO: 1 that does not include an N-terminal methionine.

DTVLS variant inhibits protein synthesis D mutates the VLS motif so that the recognized x (D) y motif is destroyed
A variant of the T382 gene was generated. The activity of variants at single and multiple loci was evaluated for activity in in vitro transcription / translation assays using PCR products (as described in Example 1).

FIG. 25 shows the VL of DT382 compared to wild type DT382 in inhibiting protein synthesis.
Represents the relative activity of the S variant. Data are for the following VLS variants: V7N V29N
I290N, V7N V29N I290T, V7N V29N S292A, V7N
V29N S292T, V7N V29T I290N, V7N V29T I290T
, V7N V29T S292A and V7N V29T S292T all show equivalent activity to wild type DT382 in inhibiting protein synthesis.

Binding of VLS variants to HUVEC Human vascular endothelial cells (HUVEC) were isolated from EBM (CC-3124 Lonza, Base).
l, Switzerland). Prior to use, cells are separated from plastic culture substrates using enzyme-free dissociation buffer (C5914 Sigma, Poole, UK) and phosphate buffer containing 1% BSA and 0.05% NaN _3. Resuspended in saline. The cells are then incubated for 20 minutes in the same buffer containing 5% normal human serum, followed by Alexa488 fluorescent dye (A30006 Invitrogen,
Purified DT382 protein or DT382 complexed to Carlsbad CA)
VLS variant titrants were added according to the manufacturer's instructions. Cells are incubated with the labeled protein for 30 minutes, then washed and washed with PBS + 1% BSA +
Resuspended in 0.05% NaN ₃ buffer. Labeled DT-389-IL2 fusion was used as a positive control and labeled BSA was used as a negative control. The cells are then FACSCali
Bur flow cytometer (Becton Dickinson, Franklin)
Lakes, NJ) and fluorescent staining of the cell population was measured. The percentage of cells that showed staining above background was then plotted against the concentration of labeled protein used.

FIG. 26 represents binding of labeled DT382 VLS variant to HUVEC cells.
Labeled DT389-IL2 fusion (ONTAK®) was used as a positive control and labeled BSA was used as a negative control. The data show that purified Alexa488-labeled DT382 and DT389-IL2 (positive control) bind to HUVEC at similar levels. In contrast, VLS variant, V7N V29T S292A (shown), V7N
The binding of V29T I290N (shown) and V7N V29N I290N (not shown) shows a reduced level of binding to HUVEC compared to DT382 or DT389-IL2.

The sequence of DT-derived peptides was analyzed using Antitope iTope ™ software to predict possible MHC class II binding peptides. iTope ™
The software combines the data from the in vitro MHC class II binding test with a large database alignment of peptides that have been demonstrated to be T cell epitopes by in vitro T cell assays into a scoring matrix. Overlapped 13-mer peptides were selected using iTope ™ in 1 amino acid increments for the entire DT sequence. Binding scores were obtained by aggregating the distribution of each amino acid within the peptide at key pocket positions as derived from the scoring matrix. Possible T
A peptide (9-mer) containing cellular epitopes was identified (underlined sequence). In this case, an average binding score was predicted to bind if> 0.6 (for the 32 MHC class II alleles tested) and> 15 MHC class II alleles (FIG. 28). Each 9-mer p1 anchor configuration is highlighted in bold, and a complete list of possible binding peptides is summarized in Table 8. For comparison, T cell epitopes identified using in vitro T cell assays are shown in boxes.

Table 8. List of predicted 9-mer sequences containing T cell epitopes using iTope ™ MHC class II binding software

Variant DT-IL2 assembly and expression In step 6, one or more lead DT-IL2 variants are generated by fusion of the lead DT variant from step 5 with the human IL2 (2-133) gene from step 3. Is done. Expression of wild type and lead DT-IL2 variants in E. coli is, for example,
The conventional method of DT-IL2 including storage and regeneration of protein aggregates in inclusion bodies is followed. The wild type and one or more lead DT-IL2 variants are then tested in cytotoxicity and VLS-related assays as described in Step 2.

Immunogenicity test of lead DT variant using EpiScreen ™ Purify the lead DT (ΔR) variant from step 5 and compare to wild type DT (ΔR) using EpiScreen ™ time-lapse T cell assay . A number of healthy donors representing social populations for HLA allotype expression are selected from the donor library.
Donors are stimulated with each protein in a separate bulk culture containing 2-4 × 10 ⁶ CD8 + T cell depleted PBMC. Duplicate samples (T blast samples) are removed from bulk cultures on days 5-8 and proliferation is assessed along with IL-2 secretion (ELISPOT). To further validate the assessment between wild-type and DT (ΔR) variants, the test population was responsive to donors from the EpiScreen ™ T cell epitope mapping study performed during stage 1 (a sufficient number provided CD8 ⁺ T cell-deficient PBMC remain).

To confirm the loss of immunogenicity in the lead DT (ΔR) variant,
Analysis of T cell immunogenicity by the EpiScreen ™ time-lapse T cell assay is performed as follows.

(I) Isolating PBMC containing physiological levels of APC and CD4 ⁺ T cells using buffy coats from healthy donors (having> 80% DRB1 allotype range for social populations);
(Ii) each donor is treated with a positive control antigen such as keyhole limpet hemocyanin (
Test against possible new antigens) or tetanus toxoid (recall antigen);
(Iii) depleting CD8 ⁺ T cells to rule out detection of MHC class I restricted T cell responses;
(Iv) Lead DT (ΔR) variants and wild type DT (ΔR) are compared against each other to assess the relative ability to activate T cell CD4 ⁺ T cells;
(V) Data is supported by additional information including statistical and frequency analysis SI>
Analyzing using previously demonstrated assay parameters with 2 positive responses;
(Vi) Data from the EpiScreen ™ time-lapse T cell assay shows individual DT
Provide information on the magnitude and kinetics of T cell responses to molecules;
(Vii) Residual PBMC from donors that produce a positive response are stored and available for use in replicate studies; and (viii) Donor allotype and response to DT (ΔR) and variants DT (
ΔR) Assess the relevance of the response to the lead.

Adjuvant effect of DT variant-IL2 fusion protein Clinical trial design and patient eligibility Treatment of patients is subject to Institutional Review Boa
rd) and written informed consent as part of a protocol approved by the US Food and Drug Administration. Patients with histologically confirmed metastatic RCC are eligible for the study. All patients are required to have adequate liver, kidney and nerve function, a lifespan of more than 6 months and a Karnovsky performance status of over 70%. Patients must have returned to normal from any toxicity associated with any previous treatment and have not received any chemotherapy, radiation therapy or immunotherapy for at least 6 weeks prior to entry into the study. Don't be. Patients with CNS metastases, patients with a history of autoimmune disease, and patients with severe concurrent chronic or acute disease are excluded from this study. Patients receiving immunosuppressants are also excluded. Eligible subjects are randomly selected with the same probability of receiving a single dose of DT variant-IL2 fusion protein (18 μg / kg) followed by immunization with tumor RNA-transfected DC or DT variant-IL2 fusion protein alone. All subjects receive three intradermal injections of tumor RNA-transfected DC. This injection consists of 1 × 10 ⁷ cells administered intradermally at 2-week intervals and suspended in 200 μl 0.9% sodium chloride with each injection. Following treatment, subjects are evaluated for clinical toxicity and immune and clinical responses. Due to regulatory limitations and limited access to tumor tissue in certain subjects, tumor biopsy is not performed.

Preparation of DT variant-IL2 fusion protein and composition DT variant-IL2 fusion protein is provided as a freeze sterilized solution formulated at a concentration of 150 μg / ml in citrate buffer in a 2 ml single use vial. After thawing, D
T variant-IL2 fusion protein is diluted to a final concentration of 15 μg / ml with sterile standard saline and delivered by intravenous infusion over 30 minutes. Patients are allowed to receive acetaminophen (600 mg) and antihistamine 30-60 minutes prior to infusion. DC
For culture, the concentrated leukocyte fraction is collected by leukocyte separation. PBMCs are isolated from leukocyte separation products by density gradient centrifugation (Histopaque; Sigma-Aldr).
ich). The semiadherent cell fraction was purified from recombinant human IL-4 (500 U / ml; R & D Sys
tems) and recombinant human GM-CSF (rhGM-CSF, 800 U / ml; Immu
nex Corp. ) Supplemented with serum-free X-VIVO15 medium (Cambrex C)
orp. ) In DC culture. After 7 days, immature DCs are collected and transferred with total RNA extracted from tumor tissue that is histologically classified as clear cell carcinoma. Control RNA used for immunological monitoring studies is isolated from autologous benign kidney tissue (RE) or PBMC. Transfer of immature DC is performed by electroporation. DCs are washed in PBS and Vi
Resuspend in aSpan (Barr Laboratories) at a concentration of 4 × 10 ⁷ cells / ml. The cells are then co-incubated with 5 μg RNA per 1 × 10 ⁶ cells for 5 minutes and electroporated by exponential decay delivery at 300 V and 150 μF in a 0.4 cm cuvette (Gene Pulser II; Bio-Rad). . After electroporation, the cells were resuspended in X-VIVO15 medium and 10 ng / ml TNF-α, 10
ng / ml IL-1β, 150 ng / ml IL-6 (R & D Systems) and 1 μg / ml prostaglandin E ₂ (PGE ₂ ; Cayman Chemical)
Co. ) In the presence of Fully mature DC: Lin ^neg before administration
To ensure that the typical phenotypes of HLA class I and II ^high , CD86 ^high and CD83 ^high are met, the cells are characterized.

Assessment of immune status Analysis of IFN-γ and IL-4ELISPOT is performed using PBMC obtained before, during and after immunization. PBMC are cultured overnight in complete RPMI 1640 medium.
From PBMC, CD4 + T cells and CD8 + T cells were negatively depleted (neg
isolated by active depletion) (Miltenyi Biotec)
). After blocking, 1 × 10 ⁵ T cells and 1 × 10 ⁴ RNA transfected DCs were 2 μg / m.
96-well nitrocellulose plates (Multiscreen-IP, Mil pre-coated with 1 IFN-γ capture antibody (Pierce Biotechnology Inc.) or IL-4 capture antibody (BD Biosciences Pharmingen)
hpore) to each well. Plates were incubated at 37 ° C. for 20 hours and each well contained biotinylated IFN-γ detection antibody (Pierce Biote).
chemistry Inc. ) Or biotinylated IL-4 antibody (BD Bioscience)
ce Pharmingen). The cells are then incubated for an additional 2 hours at room temperature and then streptavidin-alkaline phosphatase (1 μg / ml, Si
gma-Aldrich) was added and the plate was developed with substrate (KPL). After washing, spots were counted using an automated ELISPOT reader (Zeiss).

CTL assays are performed by co-culturing RNA transfected DC with autologous PBMC. Cells are restimulated once and IL-2 (20 units / ml) is added after 5 days, followed every other day. After 12 days in culture, effector cells are collected. Target cells at 100 μC
200 μl complete R using i Na ₂ [51CrO ₄ ] (PerkinElmer)
Label in PMI 1640 at 37 ° C. in 5% CO ₂ for 1 hour, ⁵¹ Cr labeled target cells,
Incubate with effector cells for 5 hours at 37 ° C. in complete RPMI 1640 medium. 50 μl of the supernatant is then collected and the release of 51Cr is measured with a scintillation counter.

For proliferation assays, purified CD3 + T cells are seeded in round bottom microplates in the presence of mRNA transfected DCs. T cells alone are used as a background control. After 4 days, each well of 1 [mu] Ci ^- adding [methyl 3 H] for a further 16 h thymidine (PerkinElmer). Thymidine incorporation is measured using a liquid scintillation counter.

The cytotoxicity of the DT variant-IL2 fusion protein is measured with an MTT assay. Cells are incubated with various DT variant-IL2 fusion proteins for 6 hours before seeding in 96 well plates at a density of 5 × 10 ³ cells / well. After 48 hours of incubation, add 20 μl of MTT from a 5 mg / ml stock. After 4 hours, formazan crystals are dissolved by adding 100 μl isopropanol / 0.1 M hydrochloric acid. The absorbance of the formazan product is measured at 570 nm with an ELISA plate reader.

Cytokine secretion by vaccine-induced CD4 + T cells was determined by human Th-1 / Th-2 cytokine kit (Cytokine Bead Array, BD Bioscience).
es Pharmingen) according to the manufacturer's instructions. Isolated CD4 + T cells are restimulated overnight using RNA transfected DCs at a ratio of 10: 1.

Four color FACS analysis was performed using the following antibodies: anti-CD4 FITC, anti-CD45RO, anti-CD45.
RA (CALTAG Laboratories), anti-CD25 PE (BD Bios
This is done using sciences Pharmingen) and anti-GITR (R & D Systems) and isotype controls (CALTAG Laboratories). CD4
Selection of + / CD25neg, CD4 + / CD25int and CD4 + / CD25high T cells is performed after antibody labeling using a BD FACSAria cell sorter. FoxP
For intracellular detection of 3, cells are permeabilized with 30 μg / ml digitonin at 4 ° C. for 45 minutes. Cells are then stained with anti-FoxP3 antibody (Abcam) and R-phycoerythrin anti-goat IgG in the presence of 10 μg / ml digitonin for 30 minutes at 4 ° C. After staining, the cells are fixed and analyzed by FACS. For intracellular CTLA-4 detection, T cells were permeabilized, fixed, and biotinylated anti-CD152 (BD Bioscience)
es Pharmingen) followed by APC-streptavidin (BD Biosc)
Stained with iences Pharmingen). A total of 1 × 10 ⁶ cells are suspended in staining buffer (PBS with 1% PCS, 2 mM EDTA and 0.1% sodium azide) and incubated with antibody at 4 ° C. for 20 minutes.

The inhibitory activity of Treg isolated from PBMCs of test subjects is analyzed as previously described (Tsaknanriis et al., 2003) before and 4 days after administration of the DT variant-anti-IL2 fusion protein. J. Neurosci.Res
. 74: 296-308). CD4 + / CD25 + T cells are isolated from the test subject's PBMC using magnetic bead separation. Cells are washed with PBS and complete RPMI 1
Resuspended in 640 medium and anti-CD3 / CD28 antibody (0.4 μg / well) (CALTAG
Laboratories) into 96 well round bottom plates pre-coated.
CD4 + / CD25− cells are applied at 2.0 × 10 ⁴ / well alone or CD4 + /
Combined with CD25 + cells in triplicate wells, 1: 2 (CD4 + / CD25: CD
4 + CD25 +). On day 5, 1 μCi of ³ H-thymidine was added to the final 16
Add for time culture. Cells are then harvested with glass fiber filters and assessed for uptake of radiolabeled thymidine.

Details of real-time PCR-type quantification of β-actin transcripts have already been described in the literature. FoxP3 mRNA transcripts are quantified using the Hs00203958 ml Taq-Man gene expression assay (Applied Biosystems) according to the protocol provided by the manufacturer. A standard curve is generated using a plasmid containing the full length FoxP3 insert.

T cell analysis before and after treatment was performed on all patients who completed immunotherapy for IFN-γE.
Perform with LISPOT. The increase in antigen-specific CD4 + T cells and CD8 + T cells after immunization is compared using Wilcoxon's matched pair signed rank test, which analyzes the null hypothesis that the rate of change in T cell responses is comparable before and after treatment. 2-sid less than 0.05
ed P values are considered statistically significant.

The present invention may be embodied in other forms or carried out in other ways without departing from the spirit of the invention or its essential characteristics. Accordingly, the disclosure herein is to be considered in all aspects as illustrative and not restrictive, and is intended to embrace all variations that fall within the equivalent meaning and scope. .

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

Claims (12)

It comprises an amino acid sequence having the full-length sequence identity of 90% or more in SEQ ID NO: 2 or 200, and at least one modified T containing substitution of one or more amino acid residues in the epitope core of at least one T-cell epitope Containing a cellular epitope core,
Substitution of the one or more amino acid residues is
(I) substitution of threonine, alanine or aspartic acid for valine at amino acid residue 97 at SEQ ID NO: 2 or 200; or
(Ii) substitution of valine at position 7 of amino acid residue of SEQ ID NO: 2 or 200 with aspartic acid, substitution of leucine at position 107 of amino acid residue with asparagine, and substitution of phenylalanine at position 124 of amino acid residue with histidine ;
And
A modified diphtheria toxin wherein the modified diphtheria toxin exhibits reduced immunogenicity compared to the unmodified diphtheria toxin.   The modified diphtheria toxin according to claim 1, wherein the modified diphtheria toxin further contains a cell-binding ligand. The modified diphtheria toxin according to claim 2 , wherein the cell-binding ligand is an antibody or antigen-binding fragment thereof, cytokine, polypeptide, hormone, growth factor or insulin. The modified diphtheria toxin according to claim 3 , comprising a cytokine , and the cytokine is interleukin 2 ( IL-2 ) or interleukin 3 ( IL-3 ) . Containing antibody, and said antibody is a monoclonal antibody, polyclonal antibody, humanized antibody, recombinant antibody, or a grafted antibody, modified diphtheria toxin of claim 3. The modified diphtheria toxin according to any one of claims 2 to 5 , further comprising a peptide linker that binds the modified diphtheria toxin and a cell-binding ligand. A pharmaceutical composition comprising the modified diphtheria toxin according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. Use of the pharmaceutical composition according to claim 7 in the preparation of a therapeutic agent for a malignant disease of a subject, characterized in that the malignant disease is a blood cancer, a solid tumor or a metastasis thereof . Use of a pharmaceutical composition according to claim 7 in the preparation of a therapeutic agent for a non-malignant disease, characterized in that the non-malignant disease is graft-versus-host disease or psoriasis. Use of the pharmaceutical composition according to claim 7 and an anticancer agent in the preparation of a therapeutic agent for a malignant disease of a subject, wherein the malignant disease is a blood cancer, a solid tumor or a metastasis thereof . The blood cancer is acute myeloid leukemia, cutaneous T cell lymphoma, relapsed / refractory T cell non-Hodgkin lymphoma, relapsed / refractory B cell non-Hodgkin lymphoma, subcutaneous adipose tissue-like T cell lymphoma, extranodal natural killer Use according to claim 8 or 10 , which is / T cell lymphoma nasal type, chronic lymphocytic leukemia, solid tumor or human T cell lymphotropic virus type 1 associated acute T cell leukemia / lymphoma. The solid tumor is skin, melanoma, lung, pancreas, breast, ovary, colon, rectum, stomach, thyroid, larynx, prostate, colorectal, head, neck, eye, mouth, organ, esophagus, breast, bone, testicle A solid tumor of a tissue or organ selected from the group consisting of: lymph, bone marrow, bone, sarcoma, kidney, sweat gland, liver, kidney, brain, digestive tract, nasopharynx, urogenital tract and muscle, or any of them Use according to claim 8 or 10 , which is a metastasis.

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